Nucleoside compounds for treating viral infections

ABSTRACT

Disclosed are compounds, compositions and methods for treating viral infections caused by a  flaviviridae  family virus, such as hepatitis C virus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. utility applicationswith Ser. Nos. 10/861,090, 10/861,311, and 10/861,219 all filed on Jun.4, 2004, each of which claim the benefit under 35 U.S.C. § 119(e) ofU.S. Provisional Patent Application Ser. No. 60/515,153 filed Oct. 27,2003. The present application also claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 60/602,815 filedAug. 18, 2004. All of the above applications are incorporated herein intheir entirety.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The invention relates to methods for preparing particular compounds fortreating viral infections in mammals mediated, at least in part, by avirus in the flaviviridae family of viruses. This invention is alsodirected to novel intermediates utilized in these methods.

REFERENCES

The following publications are cited in this application as superscriptnumbers:

-   1. Giangaspero, et al., Arch. Virol. Suppl., 7: 53-62 (1993);-   2. Giangaspero, et al., Int. J. STD. AIDS, 4(5): 300-302 (1993);-   3. Yolken, et al., Lancet, 1(8637): 517-20 (1989);-   4. Wilks, et al., Lancet, 1(8629): 107 (1989);-   5. Giangaspero, et al., Lancet, 2: 110 (1988);-   6. Potts, et al., Lancet, 1(8539): 972-973 (1987);-   7. Cornberg, et al., “Hepatitis C: therapeutic perspectives.” Forum    (Genova), 11(2):154-62 (2001);-   8. Dymock, et al., Antivir. Chem. Chemother. 11(2):79-96 (2000);-   9. Devos, et al., International Patent Application Publication No.    WO 02/18404 A2, published 7 March, 2002;-   10. Sommadossi, et al., International Patent Application Publication    No. WO 01/90121, published 23 May, 2001;-   11. Carroll, S. S., et al., International Patent Application    Publication No. WO 02057287, published 25 July, 2002;-   12. Carroll, S. S., et al., International Patent Application    Publication No. WO 02057425, published 25 July, 2002;-   13. Roberts, et al., U.S. patent application Ser. No. 10/861,090,    filed Jun. 4, 2004.-   14. Roberts, et al., U.S. patent application Ser. No. 10/861,311,    filed Jun. 4, 2004.

All of the above publications and applications are herein incorporatedby reference in their entirety to the same extent as if each individualpublication or application was specifically and individually indicatedto be incorporated by reference in its entirety.

2. State of the Art

The Flaviviridae family of viruses is composed of three genera:pestivirus, flavivirus and hepacivirus (hepatitis C virus). Of thesegenera, flaviviruses and hepaciviruses represent important pathogens ofman and are prevalent throughout the world. There are 38 flavivirusesassociated with human disease, including the dengue fever viruses,yellow fever virus and Japanese encephalitis virus. Flaviviruses cause arange of acute febrile illnesses and encephalitic and hemorrhagicdiseases. Hepaciviruses currently infect approximately 2 to 3% of theworld population and cause persistent infections leading to chronicliver disease, cirrhosis, hepatocellular carcinoma and liver failure.Human pestiviruses have not been as extensively characterized as theanimal pestiviruses. However, serological surveys indicate considerablepestivirus exposure in humans. Pestivirus infections in man have beenimplicated in several diseases including, but not likely limited to,congenital brain injury, infantile gastroenteritis and chronic diarrheain human immunodeficiency virus (HIV) positive patients. ¹⁻⁶

Currently, there are no antiviral pharmaceutical drugs to prevent ortreat pestivirus or flavivirus infections. For hepacivirus, i.e.,hepatitis C virus (HCV) infections, interferon alpha (IFN) is currentlythe only approved drug in the United States. HCV is a major causativeagent for post-transfusion and for sporadic non-A, non-B hepatitis.Infection by HCV is insidious in a high proportion of chronicallyinfected (and infectious) carriers who may not experience clinicalsymptoms for many years.

At present, the only acceptable treatment for chronic HCV is interferon(IFN-alpha) and this requires at least six (6) months of treatmentand/or ribavirin, which can inhibit viral replication in infected cellsand also improve liver function in some people.

IFN-alpha belongs to a family of naturally occurring small proteins withcharacteristic biological effects such as antiviral, immunoregulatoryand antitumoral activities that are produced and secreted by most animalnucleated cells in response to several diseases, in particular viralinfections. IFN-alpha is an important regulator of growth anddifferentiation affecting cellular communication and immunologicalcontrol. Treatment of HCV with interferon, however, has limited longterm efficacy with a response rate about 25%. In addition, treatment ofHCV with interferon has frequently been associated with adverse sideeffects such as fatigue, fever, chills, headache, myalgias, arthralgias,mild alopecia, psychiatric effects and associated disorders, autoimmunephenomena and associated disorders and thyroid dysfunction.

Ribavirin (1-β-D-ribofuranosyl-1H-1,2,-4-triazole-3-carboxamide), aninhibitor of inosine 5′-monophosphate dehydrogenase (IMPDH), enhancesthe efficacy of IFN-alpha in the treatment of HCV. Despite theintroduction of Ribavirin, more than 50% of the patients do noteliminate the virus with the current standard therapy ofinterferon-alpha (IFN) and Ribavirin. By now, standard therapy ofchronic hepatitis C has been changed to the combination of PEG-IFN plusribavirin. However, a number of patients still have significant sideeffects, primarily related to Ribavirin. Ribavirin causes significanthemolysis in 10-20% of patients treated at currently recommended doses,and the drug is both teratogenic and embryotoxic.

Other approaches are being taken to combat the virus. They include, forexample, application of antisense oligonucleotides or ribozymes forinhibiting HCV replication. Furthermore, low-molecular weight compoundsthat directly inhibit HCV proteins and interfere with viral replicationare considered as attractive strategies to control HCV infection. NS3/4Aserine protease, ribonucleic acid (RNA) helicase, RNA-dependent RNApolymerase are considered as potential targets for new drugs.^(7,8)

Devos, et al.⁹ describes purine and pyrimidine nucleoside derivativesand their use as inhibitors of HCV RNA replication. Sommadossi, et al.¹⁰describes 1′, 2′ or 3′-modified nucleosides and their use for treating ahost infected with HCV. Carroll, et al.^(11,12), describes nucleosidesas inhibitors of RNA-dependent RNA viral polymerase.

Recently, Roberts, et al.^(13,14) disclosed that certain7-(2′-substituted-β-D-ribofuranosyl)-4-amino-5-(optionally substitutedethyn-1-yl)-pyrrolo[2,3-d]pyrimidine compounds possess potent activityagainst HCV. These references are incorporated herein by reference intheir entirety.

SUMMARY OF THE INVENTION

This invention is directed to novel compounds that are useful in thetreatment of viral infections in mammals mediated at least in part by avirus in the flaviviridae family of viruses. Specifically this inventionis directed to compounds of Formula I

wherein

-   Y is selected from the group consisting of a bond, —CH₂— or —O—;-   each of W, W¹ and W² is independently selected from the group    consisting of hydrogen and a pharmaceutically acceptable prodrug;    and-   T is selected from the group consisting of-   a) —C≡C—R, where R is selected from the group consisting of    -   i) tri(C₁-C₄)alkylsilyl, —C(O)NR¹R², alkoxyalkyl, heteroaryl,        substituted heteroaryl, phenyl, and phenyl substituted with 1 to        3 substituents selected from the group consisting of alkyl,        substituted alkyl, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, alkoxy, substituted alkoxy, acyl,        acylamino, acyloxy, aminoacyl, amidino, amino, substituted        amino, carboxyl, carboxyl ester, cyano, cycloalkyl, substituted        cycloalkyl, cycloalkoxy, substituted cycloalkoxy, guanidino,        halo, heteroaryl, substituted heteroaryl, hydrazino, hydroxyl,        nitro, thiol, and —S(O)_(m)R³;    -   where R¹ and R² are independently selected from the group        consisting of hydrogen, alkyl, substituted alkyl, amino,        substituted amino, aryl, substituted aryl, heteroaryl,        substituted heteroaryl, heterocyclic and substituted        heterocyclic provided that only one of R¹ and R² is amino or        substituted amino, and further wherein R¹ and R², together with        the nitrogen atom pendant thereto, form a heterocyclic or        substituted heterocyclic;    -   R³ is selected from the group consisting of alkyl, substituted        alkyl, amino, substituted amino, aryl, substituted aryl,        heteroaryl, substituted heteroaryl, heterocyclic and substituted        heterocyclic; and    -   m is an integer equal to 0, 1 or 2;    -   ii) —C(O)OR¹⁴, where R¹⁴ is hydrogen, alkyl or substituted        alkyl;-   b) —CH═CH-Q², where Q² is selected from hydrogen or cis-alkoxy;-   c) —C(O)H;-   d) —CH═NNHR¹⁵, where R¹⁵ is H or alkyl;-   e) —CH═N(OR¹⁵), where R¹⁵ is as defined above;-   f) —CH(OR¹⁶)₂, where R₁₆ is (C₃-C₆)alkyl; and-   g) —B(OR¹⁵)₂, where R¹⁵ is as defined above;    and pharmaceutically acceptable salts or partial salts thereof.

In one preferred embodiment T is —C≡C—R and R is selected from the groupconsisting of tri(C₁-C₄)alkylsilyl, —C(O)NR¹R², alkoxyalkyl, heteroaryl,substituted heteroaryl, phenyl, and phenyl substituted with 1 to 3substituents selected from the group consisting of alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, aminoacyl,amidino, amino, substituted amino, carboxyl, carboxyl ester, cyano,cycloalkyl, substituted cycloalkyl, cycloalkoxy, substitutedcycloalkoxy, guanidino, halo, heteroaryl, substituted heteroaryl,hydrazino, hydroxyl, nitro, thiol, and —S(O)_(m)R³;

-   where R¹ and R² are independently selected from the group consisting    of hydrogen, alkyl, substituted alkyl, amino, substituted amino,    aryl, substituted aryl, heteroaryl, substituted heteroaryl,    heterocyclic and substituted heterocyclic provided that only one of    R¹ and R² is amino or substituted amino, and further wherein R¹ and    R², together with the nitrogen atom pendant thereto, form a    heterocyclic or substituted heterocyclic;-   R³ is selected from the group consisting of alkyl, substituted    alkyl, amino, substituted amino, aryl, substituted aryl, heteroaryl,    substituted heteroaryl, heterocyclic and substituted heterocyclic;    and-   m is an integer equal to 0, 1 or 2;-   or a pharmaceutically acceptable salt thereof.-   More preferably, R is selected from the group consisting of phenyl,    —C(O)NH₂, —Si(CH₃)₃, pyrid-2-yl, 4-methoxyphenyl, and —CH(OCH₂CH₃)₂.

In another preferred embodiment T is —C≡C—R and R is —C(O)OH.

In another preferred embodiment T is —C≡C—R, R is —C(O)OR¹⁴, and R¹⁴ isalkyl.

In another preferred embodiment T is —CH═CH-Q², where Q² is selectedfrom hydrogen or cis-methoxy.

In another preferred embodiment T is —C(═O)H.

In another preferred embodiment T is —CH═NNHR¹⁵ where R¹⁵ isindependently selected from the group consisting of hydrogen and alkyl.

In another preferred embodiment T is —CH═N(OR¹⁵) where R¹⁵ isindependently selected from the group consisting of hydrogen and alkyl.

In another preferred embodiment T is —CH(OR¹⁶)₂ where R¹⁶ isindependently (C₃-C₆)alkyl.

In another preferred embodiment T is —B(OR¹⁵)₂, where R¹⁵ isindependently selected from the group consisting of hydrogen and alkyl.

In another preferred embodiment each of W, W¹, and W² is independentlyhydrogen or a pharmaceutically acceptable prodrug selected from thegroup consisting of acyl, oxyacyl, phosphonate, phosphate esters,phosphate, phosphonamidate, phosphorodiamidate, phosphoramidatemonoester, cyclic phosphoramidate, cyclic phosphorodiamidate,phosphoramidate diester, and —C(O)CHR³⁰NH₂ where R³⁰ is selected fromthe group consisting of hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl and substituted heteroaryl. Preferably R³⁰is a sidechain of an amino acid and more preferably is derived from anL-amino acid.

Preferably, W is H, or W¹ is H, or W² is H. More preferably, W and W¹are H, or W and W² are H, or W¹ and W are H. Even more prefereably, W,W¹ and W² are H.

In still another preferred embodiment W¹ and W² are hydrogen and W ishydrogen or a pharmaceutically acceptable prodrug selected from thegroup consisting of acyl, oxyacyl, phosphonate, phosphate esters,phosphate, phosphonamidate, phosphorodiamidate, phosphoramidatemonoester, cyclic phosphoramidate, cyclic phosphorodiamidate,phosphoramidate diester, and —C(O)CHR³⁰NH₂.

In one particularly preferred embodiment, W¹ and W² are hydrogen and Wis represented by the formula:

where R³⁰ is as defined above, R⁸ is hydrogen or alkyl and R¹⁰ isselected from the group consisting of alkyl, substutituted alkyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic. In apreferred embodiment R³⁰ is derived from an L-amino acid.

In another particularly preferred embodiment, W and W² are hydrogen andW¹ is represented by the formula:

where R³⁰ is as defined above. As before, R³⁰ is preferably derived froman L amino acid. TABLE I # Structure Name 101

7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-(2′-trimethylsilylethyn-1-yl)- pyrrolo[2,3-d]pyrimidine 102

7-(2′-C-methyl-β-D-ribofuranosyl)-4- amino-5-[2-(pyrid-2-yl)ethyn-1-yl]-pyrrolo[2,3-d]pyrimidine 103

7-(2′-C-methyl-β-D-ribofuranosyl)-4- amino-5-[2-(pyrid-4-yl)ethyn-1-yl]-pyrrolo[2,3-d]pyrimidine 104

7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-(2-(4-methoxyphenyl)ethyn-1- yl)-pyrrolo[2,3-d]pyrimidine 105

7-(2′-C-methyl-β-D-ribofuranosyl)-4- amino-5-(2-carboxamidoethyn-1-yl)-pyrrolo[2,3-d]pyrimidine 106

7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-[(3,3-diethyl)proparg-1-yl]- pyrrolo[2,3-d]pyrimidine 107

7-(2′-C-methyl-β-D-ribofuranosyl)-4- amino-5-[(N,N-dimethyl-2-carboxamido)ethyn-1-yl]-pyrrolo[2,3- d]pyrimidine 108

7-(2′-C-methyl-β-D-ribofuranosyl)-4- amino-5-[(N-amino-2-carboxamido)ethyn-1-yl]-pyrrolo[2,3- d]pyrimidine 109

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-(2′-trimethylsilylethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 110

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-[2-(pyrid-2-yl)ethyn-1-yl]-pyrrolo[2,3-d]pyrimidine 111

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-[2-(pyrid-4-yl)ethyn-1-yl]-pyrrolo[2,3-d]pyrimidine 112

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-(2-(4-methoxyphenyl)ethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 113

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-(2-carboxamidoethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 114

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-[(3,3-diethoxy)proparg-1-yl]-pyrrolo[2,3- d]pyrimidine 115

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-[2-(N,N-dimethylcarboxamido)ethyn-1-yl]- pyrrolo[2,3-d]pyrimidine 116

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-[2-(N-aminocarboxamido)ethyn-1-yl]- pyrrolo[2,3-d]pyrimidine 117

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-(2′-trimethylsilylethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 118

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-[2-(pyrid-2-yl)ethyn-1-yl]-pyrrolo[2,3-d]pyrimidine 119

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-[2-(pyrid-4-yl)ethyn-1-yl]-pyrrolo[2,3-d]pyrimidine 120

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-(2-(4-methoxyphenyl)ethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 121

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-(2-carboxamidoethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 122

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-[(3,3-diethoxy)proparg-1-yl]-pyrrolo[2,3- d]pyrimidine 123

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-[(N,N-dimethyl-2-carboxamido)ethyn-1-yl]- pyrrolo[2,3-d]pyrimidine 124

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-[(N-amino-2-carboxamido)ethyn-1-yl]-pyrrolo[2,3- d]pyrimidine 125

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-(2′-trimethylsilylethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 126

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-[2-(pyrid-2-yl)ethyn-1-yl)-pyrrolo[2,3-d]pyrimidine 127

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-[2-(pyrid-2-yl)ethyn-1-yl)-pyrrolo[2,3-d]pyrimidine 128

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-(2-(4-methoxyphenyl)ethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 129

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-(2-carboxamidoethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 130

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-[(3,3-diethoxy)proparg-1-yl]-pyrrolo[2,3- d]pyrimidine 131

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-[(N,N-dimethylcarboxylamido)ethyn-1-yl]- pyrrolo[2,3-d]pyrimidine 132

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-[(N-aminocarboxylamido)ethyn-1-yl]- pyrrolo[2,3-d]pyrimidine 133

7-(2′-C-methyl-β-D-ribofuranosyl)-4- amino-5-(2-carboxyethyn-1-yl)-pyrrolo[2,3-d]pyrimidine 134

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-(2-carboxyethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 135

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-(2-carboxyethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 136

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-(2-carboxyethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 137

7-(2′-C-methyl-β-D-ribofuranosyl)-4- amino-5-[(ethyl2-carboxyl)ethyn-1-yl]- pyrrolo[2,3-d]pyrimidine 138

7-(2′-C-methyl-β-D-ribofuranosyl)-4- amino-5-[(methyl2-carboxyl)ethyn-1- yl]-pyrrolo[2,3-d]pyrimidine 139

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-[(ethyl 2-carboxyl)ethyn-1-yl]-pyrrolo[2,3- d]pyrimidine 140

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-[(methyl 2-carboxyl)ethyn-1-yl]-pyrrolo[2,3- d]pyrimidine 141

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-[(ethyl 2-carboxyl)ethyn-1-yl]-pyrrolo[2,3- d]pyrimidine 142

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-[(methyl 2-carboxyl)ethyn-1-yl]-pyrrolo[2,3- d]pyrimidine 143

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-[(ethyl 2-carboxyl)ethyn-1-yl]-pyrrolo[2,3- d]pyrimidine 144

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-[(methyl 2-carboxyl)ethyn-1-yl]-pyrrolo[2,3- d]pyrimidine 145

7-(2′-C-methyl-β-D-ribofuranosyl)-4- amino-5-(2-phenylethyn-1-yl)-pyrrolo[2,3-d]pyrimidine 146

7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-(2-(4-fluorophenyl)ethyn-1-yl)- pyrrolo[2,3-d]pyrimidine 147

7-(2′-C-methyl-β-D-ribofuranosyl)-4- amino-5-(2-(4-methylphenyl)ethyn-1-yl)-pyrrolo[2,3-d]pyrimidine 148

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-(2-phenylethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 149

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-(2-(4-fluorophenyl)ethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 150

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-(2-(4-methylphenyl)ethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 151

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-(2-phenylethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 152

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-(2-(4-fluorophenyl)ethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 153

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-(2-(4-methylphenyl)ethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 154

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-(2-phenylethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 155

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-(2-(4-fluorophenyl)ethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 156

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-(2-(4-methylphenyl)ethyn-1-yl)-pyrrolo[2,3- d]pyrimidine 157

7-(2′-C-methyl-β-D-ribofuranosyl)-4- amino-5-(ethen-1-yl)-pyrrolo[2,3-d]pyrimidine 158

7-(2′-C-methyl-β-D-ribofuranosyl)-4- amino-5-(2-cis-methoxy-ethen-1-yl)-pyrrolo[2,3-d]pyrimidine 159

7-(2′-C-methyl-5′-monophospho-β-D-ribofuranosyl)-4-amino-5-(ethen-1-yl)- pyrrolo[2,3-d]pyrimidine 160

7-(2′-C-methyl-5′-monophospho-β-D- ribofuranosyl)-4-amino-5-(2-cis-methoxy-ethen-1-yl)-pyrrolo[2,3- d]pyrimidine 161

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-(ethen-1-yl)-pyrrolo[2,3-d]pyrimidine 162

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-(2-cis-methoxy-ethen-1-yl)-pyrrolo[2,3- d]pyrimidine 163

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-(ethen-1-yl)-pyrrolo[2,3-d]pyrimidine 164

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-(2-cis-methoxy-ethen-1-yl)-pyrrolo[2,3- d]pyrimidine 165

7-(2′-C-methyl-β-D-ribofuranosyl)-4- amino-5-(formyl)-pyrrolo[2,3-d]pyrimidine 166

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-(formyl)-pyrrolo[2,3-d]pyrimidine 167

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-(formyl)-pyrrolo[2,3-d]pyrimidine 168

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-(formyl)-pyrrolo[2,3-d]pyrimidine 169

7-(2′-C-methyl-β-D-ribofuranosyl)-4- amino-5-(hydrazono)-pyrrolo[2,3-d]pyrimidine 170

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-(hydrazono)-pyrrolo[2,3-d]pyrimidine 171

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-(hydrazono)-pyrrolo[2,3-d]pyrimidine 172

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-(hydrazono)-pyrrolo[2,3-d]pyrimidine 173

7-(2′-C-methyl-β-D-ribofuranosyl)-4- amino-5-(carbaldehyde oxime)-pyrrolo[2,3-d]pyrimidine 174

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-(carbaldehydeoxime)-pyrrolo[2,3-d]pyrimidine 175

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-(carbaldehydeoxime)-pyrrolo[2,3-d]pyrimidine 176

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-(carbaldehydeoxime)-pyrrolo[2,3-d]pyrimidine 177

7-(2′-C-methyl-β-D-ribofuranosyl)-4- amino-5-(diisopropoxymethyl)-pyrrolo[2,3-d]pyrimidine 178

7-(2′-C-methyl-5′-monophospho-β-D- ribofuranosyl)-4-amino-5-(diisopropoxymethyl)-pyrrolo[2,3- d]pyrimidine 179

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-(diisopropoxymethyl)-pyrrolo[2,3- d]pyrimidine 180

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-(diisopropoxymethyl)-pyrrolo[2,3- d]pyrimidine 181

7-(2′-C-methyl-β-D-ribofuranosyl)-4- amino-5-(boronic acid)-pyrrolo[2,3-d]pyrimidine 182

7-(2′-C-methyl-5′-phospho-β-D- ribofuranosyl)-4-amino-5-(boronic acid)-pyrrolo[2,3-d]pyrimidine 183

7-(2′-C-methyl-5′-diphospho-β-D- ribofuranosyl)-4-amino-5-(boronicacid)-pyrrolo[2,3-d]pyrimidine 184

7-(2′-C-methyl-5′-triphospho-β-D- ribofuranosyl)-4-amino-5-(boronicacid)-pyrrolo[2,3-d]pyrimidine

Compounds of this invention are either active as antiviral agents or areuseful as intermediates in the preparation of antiviral agents asdescribed herein.

This invention is also directed to pharmaceutical compositionscomprising a pharmaceutically acceptable diluent and a therapeuticallyeffective amount of a compound as described herein or mixtures of one ormore of such compounds.

This invention is still further directed to methods for treating a viralinfection mediated at least in part by a virus in the flaviviridaefamily of viruses, such as HCV, in mammals which methods compriseadministering to a mammal, that has been diagnosed with said viralinfection or is at risk of developing said viral infection, apharmaceutical composition comprising a pharmaceutically acceptablediluent and a therapeutically effective amount of a compound asdescribed herein or mixtures of one or more of such compounds.

In yet another embodiment of the invention, methods of treating orpreventing viral infections in mammals are provided where in thecompounds of this invention are administered in combination with theadministration of a therapeutically effective amount of one or moreagents active against HCV. Active agents against HCV include ribavirin,levovirin, viramidine, thymosin alpha-1, an inhibitor of NS3 serineprotease, and inhibitor of inosine monophosphate dehydrogenase,interferon-alpha, pegylated interferon-alpha, alone or in combinationwith ribavirin or levovirin. Preferably the additional agent activeagainst HCV is interferon-alpha or pegylated interferon-alpha alone orin combination with ribavirin or levovirin.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to compounds, compositions and methods fortreating flaviviridae viruses, such as hepatitis C virus infections.However, prior to describing this invention in detail, the followingterms will first be defined:

Definitions

As used herein, the term “alkyl” refers to alkyl groups having from 1 to6 carbon atoms, preferably 1 to 3, and more preferably 1 to 2 carbonatoms. This term is exemplified by groups such as methyl, ethyl,n-propyl, iso-propyl, n-butyl, t-butyl, n-pentyl and the like.

“Substituted alkyl” refers to an alkyl group having from 1 to 3, andpreferably 1 to 2, substituents selected from the group consisting ofalkoxy, substituted alkoxy, acyl, acylamino, acyloxy, oxyacyl, amino,substituted amino, aminoacyl, aryl, substituted aryl, aryloxy,substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl, carboxylesters, cycloalkyl, substituted cycloalkyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic.

“Alkoxy” refers to the group “alkyl-O—” which includes, by way ofexample, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, t-butoxy,sec-butoxy, n-pentoxy and the like.

“Substituted alkoxy” refers to the group “substituted alkyl-O—”.

“Alkoxyalkyl” refers to the groups -alkylene(alkoxy)_(n)-alkylene(substituted alkoxy)_(n) where alkylene is a divalent straightor branched chain alkylene group of from 1 to 3 carbon atoms, alkoxy andsubstituted alkoxy is as defined herein and n is an integer from 1 to 2.

“Acyl” refers to the groups alkyl-C(O)—, substituted alkyl-C(O)—,alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—, substitutedalkynyl-C(O)— cycloalkyl-C(O)—, substituted cycloalkyl-C(O)—,aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, substitutedheteroaryl-C(O), heterocyclic-C(O)—, and substituted heterocyclic-C(O)—.

“Acylamino” refers to the group —C(O)NR⁴R⁴ where each R⁴ isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic and where each R⁴ is joined to form together with thenitrogen atom a heterocyclic or substituted heterocyclic ring.

“Acyloxy” refers to the groups alkyl-C(O)O—, substituted alkyl-C(O)O—,alkenyl-C(O)O—, substituted alkenyl-C(O)O—, alkynyl-C(O)O—, substitutedalkynyl-C(O)O—, aryl-C(O)O—, substituted aryl-C(O)O—, cycloalkyl-C(O)O—,substituted cycloalkyl-C(O)O—, heteroaryl-C(O)O—, substitutedheteroaryl-C(O)O—, heterocyclic-C(O)O—, and substitutedheterocyclic-C(O)O—.

“Oxyacyl” refers to the groups alkyl-OC(O)—, substituted alkyl-OC(O)—,alkenyl-OC(O)—, substituted alkenyl-OC(O)—, alkynyl-OC(O)—, substitutedalkynyl-OC(O)—, aryl-OC(O)—, substituted aryl-OC(O)—, cycloalkyl-OC(O)—,substituted cycloalkyl-OC(O)—, heteroaryl-OC(O)—, substitutedheteroaryl-OC(O)—, heterocyclic-OC(O)—, and substitutedheterocyclic-OC(O)—.

“Alkenyl” refers to alkenyl group preferably having from 2 to 6 carbonatoms and more preferably 2 to 4 carbon atoms and having at least 1 andpreferably from 1-2 sites of alkenyl unsaturation. Such groups areexemplified by vinyl (ethen-1-yl), allyl, but-3-en-1-yl, and the like.

“Substituted alkenyl” refers to alkenyl groups having from 1 to 3substituents, and preferably 1 to 2 substituents, selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl,aryloxy, substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl,carboxyl esters, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic withthe proviso that any hydroxyl substitution is not attached to a vinyl(unsaturated) carbon atom. Preferred substituted alkenyl groups areselected from, but not limit to, 2,2-difluoroethen-1-yl,2-methoxyethen-1-yl, and the like.

It is understood that the term “substituted alkenyl” includes both E(cis) and Z (trans) isomers as appropriate. The isomers can be pureisomeric compounds or mixtures of E and Z components.

“Alkynyl” refers to an unsaturated hydrocarbon having at least 1 site ofalkynyl unsaturation and having from 2 to 6 carbon atoms and morepreferably 2 to 4 carbon atoms. Preferred alkynyl groups are selectedfrom but not limit to ethyn-1-yl, propyn-1-yl, propyn-2-yl,1-methylprop-2-yn-1-yl, butyn-1-yl, butyn-2-yl, butyn-3-yl, and thelike.

“Substituted alkynyl” refers to alkynyl groups having from 1 to 3substituents, and preferably 1 to 2 substituents, selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl,aryloxy, substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl,carboxyl esters, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic.Preferred substituted alkynyl groups are selected from but not limit to2-fluoroethyn-1-yl, 3,3,3-trifluoropropyn-1-yl, 3-aminopropyn-1-yl,3-hydroxypropyn-1-yl, and the like.

“Amino” refers to the group —NH₂.

“Substituted amino” refers to the group —NR′R″ where R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic and where R′ and R″ are joined, together with the nitrogenbound thereto to form a heterocyclic or substituted heterocyclic groupprovided that R′ and R″ are both not hydrogen. When R′ is hydrogen andR″ is alkyl, the substituted amino group is sometimes referred to hereinas alkylamino. When R′ and R″ are alkyl, the substituted amino group issometimes referred to herein as dialkylamino.

“Amidino” refers to the group —C(═NR¹¹)NR¹¹R¹¹ where each R¹¹ isindependently selected from hydrogen or alkyl.

“Aminoacyl” refers to the groups —NR⁵C(O)alkyl, —NR⁵C(O)substitutedalkyl, —NR⁵C(O)cycloalkyl, —NR⁵C(O)substituted cycloalkyl,—NR⁵C(O)alkenyl, —NR⁵C(O)substituted alkenyl, —NR⁵C(O)alkynyl,—NR⁵C(O)substituted alkynyl, —NR⁵C(O)aryl, —NR⁵C(O)substituted aryl,—NR⁵C(O)heteroaryl, —NR⁵C(O)substituted heteroaryl,—NR⁵C(O)heterocyclic, and —NR⁵C(O)substituted heterocyclic where R⁵ ishydrogen or alkyl.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl) which condensed rings may ormay not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the pointof attachment is at an aromatic carbon atom. Preferred aryls includephenyl and naphthyl.

“Substituted aryl”, including “substituted phenyl” refers to aryl groupsor phenyl groups which are substituted with from 1 to 3 substituents,and preferably 1 to 2 substituents, selected from the group consistingof hydroxyl, acyl, acylamino, acyloxy, alkyl, substituted alkyl, alkoxy,substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, amino, substituted amino, aminoacyl, aryl, substituted aryl,aryloxy, substituted aryloxy, cycloalkoxy, substituted cycloalkoxy,carboxyl, carboxyl esters, cyano, thiol, thioalkyl, substitutedthioalkyl, thioaryl, substituted thioaryl, thioheteroaryl, substitutedthioheteroaryl, thiocycloalkyl, substituted thiocycloalkyl,thioheterocyclic, substituted thioheterocyclic, cycloalkyl, substitutedcycloalkyl, halo, nitro, heteroaryl, substituted heteroaryl,heterocyclic, substituted heterocyclic, heteroaryloxy, substitutedheteroaryloxy, heterocyclyloxy, and substituted heterocyclyloxy.

“Aryloxy” refers to the group aryl-O— that includes, by way of example,phenoxy, naphthoxy, and the like.

“Substituted aryloxy” refers to substituted aryl-O— groups.

“Carboxyl” refers to —COOH or salts thereof.

“Carboxyl esters” refers to the groups —C(O)O-alkyl, —C(O)O-substitutedalkyl, —C(O)Oaryl, and —C(O)O-substituted aryl wherein alkyl,substituted alkyl, aryl and substituted aryl are as defined herein.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including, by way of example,adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and thelike.

“Substituted cycloalkyl” refers to a cycloalkyl having from 1 to 5substituents selected from the group consisting of oxo (═O), thioxo(═S), alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aryl,substituted aryl, aryloxy, substituted aryloxy, cyano, halogen,hydroxyl, nitro, carboxyl, carboxyl esters, cycloalkyl, substitutedcycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic.

“Cycloalkoxy” refers to —O-cycloalkyl groups.

“Substituted cycloalkoxy” refers to —O-substituted cycloalkyl groups.

“Formyl” refers to the group —C(O)H.

“Guanidino” refers to the group —NR¹²C(═NR¹²)NR²R¹² where each R¹² isindependently hydrogen or alkyl.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo andpreferably is fluoro or chloro.

“Heteroaryl” refers to an aromatic group of from 1 to 10 carbon atomsand 1 to 4 heteroatoms selected from the group consisting of oxygen,nitrogen, and sulfur within the ring wherein the nitrogen and/or sulfuris optionally oxidized [(N→O), —S(O)—, or —SO₂—]. Such heteroaryl groupscan have a single ring (e.g., pyridyl or furyl) or multiple condensedrings (e.g., indolizinyl or benzothienyl) wherein the condensed ringsmay or may not be aromatic and/or contain a heteroatom provided that thepoint of attachment is through an aromatic ring atom. Preferredheteroaryls include pyridyl, pyrrolyl, indolyl, thiophenyl, and furyl.

“Substituted heteroaryl” refers to heteroaryl groups that aresubstituted with from 1 to 3 substituents selected from the same groupof substituents defined for substituted aryl.

“Heteroaryloxy” refers to the group —O-heteroaryl and “substitutedheteroaryloxy” refers to the group —O-substituted heteroaryl.

“Heterocycle” or “heterocyclic” or “heterocycloalkyl” refers to asaturated or unsaturated group (but not heteroaryl) having a single ringor multiple condensed rings, from 1 to 10 carbon atoms and from 1 to 4hetero atoms selected from the group consisting of nitrogen, oxygen andsulfur within the ring wherein the nitrogen and/or sulfur atoms can beoptionally oxidized [(N→O), —S(O)— or —SO₂—]. and further wherein, infused ring systems, one or more the rings can be cycloalkyl, aryl orheteroaryl provided that the point of attachment is through theheterocyclic ring.

“Substituted heterocyclic” or “substituted heterocycloalkyl” refers toheterocycle groups that are substituted with from 1 to 3 of the samesubstituents as defined for substituted cycloalkyl.

Examples of heterocycles and heteroaryls include, but are not limitedto, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), piperidinyl, pyrrolidine, tetrahydrofuranyl, and thelike.

“Heterocyclyloxy” refers to the group —O-heterocyclic and “substitutedheterocyclyloxy” refers to the group —O-substituted heterocyclic.

“Hydrazino” refers to the group —NR¹³NR¹³R¹³ wherein each R¹³ isindependently selected from the group consisting of hydrogen or alkyl.

“Phosphate” refers to the groups —OP(O)(OH)₂ (monophosphate or phospho),—OP(O)(OH)OP(O)(OH)₂ (diphosphate or diphospho) and—OP(O)(OH)OP(O)(OH)OP(O)(OH)₂ (triphosphate or triphospho) or saltsthereof including partial salts thereof. It is understood, of course,that the initial oxygen of the mono-, di- and triphosphate (phospho,diphospho and triphospho) includes the oxygen atom at the 5-position ofthe ribose sugar.

“Phosphate esters” refers to the mono-, di- and tri-phosphate groupsdescribed above wherein one or more of the hydroxyl groups is replacedby an alkoxy group.

“Phosphonate” refers to the groups —OP(O)(R⁶)(OH) or —OP(O)(R⁶)(OR⁶) orsalts thereof including partial salts thereof, wherein each R⁶ isindependently selected from hydrogen, alkyl, substituted alkyl,carboxylic acid, and carboxyl ester. It is understood, of course, thatthe initial oxygen of the phosphonate includes the oxygen atom at the5-position of the ribose sugar.

“Phosphorodiamidate” refers to the group:

where each R⁷ may be the same or different and each is hydrogen, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl. A particularlypreferred phosphorodiamidate is the following group:

“Phosphoramidate monoester” refers to the group below, where R³⁰ is asdefined above, R⁸ is hydrogen or alkyl. In a preferred embodiment R³⁰ isderived from an L-amino acid

“Phosphoramidate diester” refers to the group below, where R³⁰ is asdefined above, R⁸ is hydrogen or alkyl and R¹⁰ is selected from thegroup consisting of alkyl, substutituted alkyl, aryl, substituted aryl,cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic. In a preferred embodiment R³⁰is derived from an L-amino acid.

“Cyclic phosphoramidate” refers to the group below, where n is 1 to 3,more preferably n is 1 to 2.

“Cyclic phosphorodiamidate” refers to the group below, where n is 1 to3, more preferably n is 1 to 2.

“Phosphonamidate” refers to the group below, where R¹⁴ is hydrogen,alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

“Thiol” refers to the group —SH.

“Thioalkyl” or “alkylthioether” or “thioalkoxy” refers to the group—S-alkyl.

“Substituted thioalkyl” or “substituted alkylthioether” or “substitutedthioalkoxy” refers to the group —S-substituted alkyl.

“Thiocycloalkyl” refers to the groups —S-cycloalkyl and “substitutedthiocycloalkyl” refers to the group —S-substituted cycloalkyl.

“Thioaryl” refers to the group —S-aryl and “substituted thioaryl” refersto the group —S-substituted aryl.

“Thioheteroaryl” refers to the group —S-heteroaryl and “substitutedthioheteroaryl” refers to the group —S-substituted heteroaryl.

“Thioheterocyclic” refers to the group —S-heterocyclic and “substitutedthioheterocyclic” refers to the group —S-substituted heterocyclic.

The term “amino acid sidechain” refers to the R³⁰ substituent of α-aminoacids of the formula NH₂CH(R³⁰)COOH where R³⁰ is selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, aryl and substitutedaryl. Preferably, the α-amino acid sidechain is the sidechain one of thetwenty naturally occurring L amino acids.

The term “pharmaceutically acceptable prodrugs” refers to art recognizedmodifications to one or more functional groups which functional groupsare metabolized in vivo to provide a compound of this invention or anactive metabolite thereof. Such functional groups are well known in theart including acyl groups for hydroxyl and/or amino substitution, estersof mono-, di- and tri-phosphates wherein one or more of the pendenthydroxyl groups have been converted to an alkoxy, a substituted alkoxy,an aryloxy or a substituted aryloxy group, and the like.

The term “pharmaceutically acceptable salt” refers to pharmaceuticallyacceptable salts of a compound, which salts are derived from a varietyof organic and inorganic counter ions well known in the art and include,by way of example only, sodium, potassium, calcium, magnesium, ammonium,tetraalkylammonium, and the like; and when the molecule contains a basicfunctionality, salts of organic or inorganic acids, such ashydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate,oxalate and the like.

The term “pharmaceutically acceptable partial salts” refers to compoundshaving a substituent capable of having more than one group form a saltbut less than the maximum amount of such groups actually form a salt.For example, a diphospho group can form a plurality of salts and, ifonly partially ionized, the resulting group is sometimes referred toherein as a partial salt.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,etc.) are not intended for inclusion herein. In such cases, the maximumnumber of such substituents is three. That is to say that each of theabove definitions is constrained by a limitation that, for example,substituted aryl groups are limited to -substituted aryl-(substitutedaryl)-substituted aryl.

Similarly, it is understood that the above definitions are not intendedto include impermissible substitution patterns (e.g., methyl substitutedwith 5 fluoro groups or a hydroxyl group alpha to ethenylic oracetylenic unsaturation). Such impermissible substitution patterns arewell known to the skilled artisan.

General Synthetic Methods

The compounds of this invention can be prepared from readily availablestarting materials using the following general methods and procedures.It will be appreciated that where typical or preferred processconditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. Suitableprotecting groups for various functional groups as well as suitableconditions for protecting and deprotecting particular functional groupsare well known in the art. For example, numerous protecting groups aredescribed in T. W. Greene and G. M. Wuts, Protecting Groups in OrganicSynthesis, Third Edition, Wiley, New York, 1999, and references citedtherein.

Furthermore, the compounds of this invention contain one or more chiralcenters. Accordingly, if desired, such compounds can be prepared orisolated as pure stereoisomers, i.e., as individual enantiomers ordiastereomers, or as stereoisomer-enriched mixtures. All suchstereoisomers (and enriched mixtures) are included within the scope ofthis invention, unless otherwise indicated. Pure stereoisomers (orenriched mixtures) may be prepared using, for example, optically activestarting materials or stereoselective reagents well-known in the art.Alternatively, racemic mixtures of such compounds can be separatedusing, for example, chiral column chromatography, chiral resolvingagents and the like.

The starting materials for the following reactions are generally knowncompounds or can be prepared by known procedures or obviousmodifications thereof. For example, many of the starting materials areavailable from commercial suppliers such as Aldrich Chemical Co.(Milwaukee, Wis., USA), Bachem (Torrance, Calif., USA), Emka-Chemce orSigma (St. Louis, Mo., USA). Others may be prepared by procedures, orobvious modifications thereof, described in standard reference textssuch as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15(John Wiley and Sons, 1991), Rodd's Chemistry of Carbon Compounds,Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989),Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March'sAdvanced Organic Chemistry, (John Wiley and Sons, 4^(th) Edition), andLarock's Comprehensive Organic Transformations (VCH Publishers Inc.,1989). Specifically, the compounds of this invention may be prepared byvarious methods known in the art of organic chemistry in general andnucleoside and nucleotide analogue synthesis in particular. Generalreviews of the preparation of nucleoside and nucleotide analoguesinclude 1) Michelson A. M. “The Chemistry of Nucleosides andNucleotides,” Academic Press, New York, 1963; 2) Goodman L. “BasicPrinciples in Nucleic Acid Chemistry,” Academic Press, New York, 1974,vol. 1, Ch. 2; and 3) “Synthetic Procedures in Nucleic Acid Chemistry,”Eds. Zorbach W. & Tipson R., Wiley, New York, 1973, vol. 1 & 2.

The synthesis of the compounds of this invention generally followseither a convergent or linear synthetic pathway as described below.

The strategies available for synthesis of compounds of this inventioninclude for example:

General Synthesis of 2′-C-Branched Nucleosides

2′-C-Branched ribonucleosides of Formula I:

where T, Y, W, W¹, and W² are as defined above, can be prepared by oneof the following general methods.

Convergent Approach: Glycosylation of Nucleobase with AppropriatelyModified Sugar

The key starting material of this process is an appropriatelysubstituted sugar with 2′-OH and 2′-H with the appropriate leavinggroup, for example, an acyl group or a chloro, bromo, fluoro or iodogroup at the 1-position. The sugar can be purchased or can be preparedby any known means including standard epimerization, substitution,oxidation and/or reduction techniques. For example, commerciallyavailable 1,3,5-tri-O-benzoyl-α-D-ribofuranose (Pfanstiel Laboratories,Inc.) can be used. The substituted sugar can then be oxidized with theappropriate oxidizing agent in a compatible solvent at a suitabletemperature to yield the 2′-modified sugar. Possible oxidizing agentsare, for example, Dess-Martin periodine reagent, Ac₂O+DCC in DMSO, Swernoxidation (DMSO, oxalyl chloride, triethylamine), Jones reagent (amixture of chromic acid and sulfuric acid), Collins's reagent(dipyridine Cr(VI) oxide, Corey's reagent (pyridinium chlorochromate),pyridinium dichromate, acid dichromate, potassium permanganate, MnO₂,ruthenium tetraoxide, phase transfer catalysts such as chromic acid orpermanganate supported on a polymer, Cl₂-pyridine, H₂O₂-ammoniummolybdate, NaBrO₂-CAN, NaOCl in HOAc, copper chromite, copper oxide,Raney nickel, palladium acetate, Meerwin-Pondorf-Verley reagent(aluminum t-butoxide with another ketone) and N-bromosuccinimide.

Coupling of an organometallic carbon nucleophile, such as a Grignardreagent, an organolithium, lithium dialkylcopper or SiMe₄ in TBAF withthe ketone with the appropriate non-protic solvent at a suitabletemperature, yields the 2′-methyl sugar. For example, CH₃MgBr/TiCl₄ orCH₃MgBr/CeCl₃ can be used as described in Wolfe et al. 1997. J. Org.Chem. 62: 1754-1759. The methylated sugar can be optionally protectedwith a suitable protecting group, preferably with an acyl, substitutedalkyl or silyl group, by methods well known to those skilled in the art,as taught by Greene et al. Protective Groups in Organic Synthesis, JohnWiley and Sons, Second Edition, 1991.

The optionally protected sugar can then be coupled to the purine base bymethods well known to those skilled in the art, as taught by TownsendChemistry of Nucleosides and Nucleotides, Plenum Press, 1994. Forexample, an acylated sugar can be coupled to a silylated base with aLewis acid, such as tin tetrachloride, titanium tetrachloride ortrimethylsilyltriflate in the appropriate solvent at a suitabletemperature. Alternatively, a halo-sugar can be coupled to a silylatedbase with the presence of trimethylsilyltriflate.

In addition to the above, the 2′-C-substituted sugars used in thesynthetic methods described herein are well known in the art and aredescribed, for example, by Sommadossi, et al.¹⁰ and by Carrol, etal.^(11,12) all of which are incorporated herein by reference in theirentirety.

Scheme 1 below describes the alternative synthesis of a protected sugarthat is useful for coupling to the bases described herein.

Formation of sugar a in Scheme 1, above, is accomplished as described byMandal, S. B., et al., Synth. Commun., 1993, 9, page 1239, starting fromcommercial D-ribose. Protection of the hydroxyl groups to form sugar bis described in Witty, D. R., et al., Tet. Lett., 1990, 31, page 4787.Sugar c and d are prepared using the method of Ning, J. et al.,Carbohydr. Res., 2001, 330, page 165, and methods described herein.Sugar e is prepared by using a modification of the Grignard reactionwith CH₃MgBr or other appropriate organometallic as described herein(with no titanium/cerium needed). Finally the halogenated sugar (X=halo)used in the subsequent coupling reaction is prepared using the sameprotection method as used in to make sugar b above. The halogenation isdescribed in Seela, U.S. Pat. No. 6,211,158.

Subsequently, any of the described nucleosides can be deprotected bymethods well known to those skilled in the art, as taught by Greene etal. Protective Groups in Organic Synthesis, Jon Wiley and Sons, SecondEdition, 1991.

An alternative approach to making protected sugars useful for couplingto heterocyclic bases is detailed in Scheme 2 below.

In Scheme 2, methylation of the hydroxyl group of compound 1 proceedsvia conventional methodology to provide for compound 2. The 2, 3 and 5hydroxyl groups of the compound 2 are each protected with2,4-dichlorobenzyl groups to provide for compound 3. Selectivedeprotection of the 2-(2′,4′-dichlorobenzyl) group on compound 3proceeds via contact with stannous chloride in a suitable solvent suchas methylene chloride, chloroform, and the like at reduced temperatures,e.g., ˜0 to 5° C., until reaction completion, e. 24-72 hours. Oxidationof the 2-hydroxyl group proceeds as described herein to provide forcompound 7. Methylation also proceeds as described herein to provide forcompound 8.

Linear Approach: Modification of a Pre-formed Nucleoside

The key starting material for this process is an appropriatelysubstituted nucleoside with a 2′-OH and 2′-H. The nucleoside can bepurchased or can be prepared by any known means including standardcoupling techniques. The nucleoside can be optionally protected withsuitable protecting groups, preferably with acyl, substituted alkyl orsilyl groups, by methods well known to those skilled in the art, astaught by Greene et al. Protective Groups in Organic Synthesis, JohnWiley and Sons, Second Edition, 1991.

The appropriately protected nucleoside can then be oxidized with theappropriate oxidizing agent in a compatible solvent at a suitabletemperature to yield the 2′-modified sugar. Possible oxidizing agentsare, for example, Dess-Martin periodine reagent, Ac₂O+DCC in DMSO, Swernoxidation (DMSO, oxalyl chloride, triethylamine), Jones reagent (amixture of chromic acid and sulfuric acid), Collins's reagent(dipyridine Cr(VI) oxide, Corey's reagent (pyridinium chlorochromate),pyridinium dichromate, acid dichromate, potassium permanganate, MnO₂ruthenium tetroxide, phase transfer catalysts such as chromic acid orpermanganate supported on a polymer, Cl₂-pyridine, H₂O₂-ammoniummolybdate, NaBrO₂-CAN, NaOCl in HOAc, copper chromite, copper oxide,Raney nickel, palladium acetate, Meerwin-Pondorf-Verley reagent(aluminum t-butoxide with another ketone) and N-bromosuccinimide.

Coupling of an organometallic carbon nucleophile, such as a Grignardreagent, an organolithium, lithium dialkylcopper or CH₃SiMe₃ in TBAFwith the ketone with the appropriate non-protic solvent at a suitabletemperature, yields the alkyl substituted nucleoside. Isolation of theappropriate isomer is conducted as needed.

Subsequently, the nucleoside can be deprotected by methods well known tothose skilled in the art, as taught by Greene et al. Protective Groupsin Organic Synthesis, John Wiley and Sons, Second Edition, 1991.

In one embodiment of the invention, the D-enantiomers are preferred.However, L-enantiomers are also contemplated to be useful herein. TheL-enantiomers corresponding to the compounds of the invention can beprepared following the same foregoing general methods, beginning withthe corresponding L-sugar or nucleoside as starting material. In aparticular embodiment, the 2′-C-branched ribonucleoside is desired.

The compounds of this invention may be prepared by various methods knownin the art of organic chemistry in general and nucleoside and nucleotideanalogue synthesis in particular. The starting materials for thesyntheses are either readily available from commercial sources or areknown or may be prepared by techniques known in the art. General reviewsof the preparation of nucleoside and nucleotide analogues are includedin the following:

-   Michelson A. M. “The Chemistry of Nucleosides and Nucleotides,”    Academic Press, New York, 1963.-   Goodman L. “Basic Principles in Nucleic Acid Chemistry,” Academic    Press, New York, 1974, vol. 1, Ch. 2.-   “Synthetic Procedures in Nucleic Acid Chemistry,” Eds. Zorbach W. &    Tipson R., Wiley, New York, 1973, vol. 1 & 2.

The 5-substituted acetylenyl-pyrrolo[2,3-d]pyrimidinyl nucleosidederivatives of the present invention can be synthesized using themethods depicted in Scheme 3 below.

A convergent approach for preparing the pyrrolo[2,3-d]pyrimidinylnucleosides is shown in Scheme 3 below. First commercially available4-chloropyrrolo[2,3-d]pyrimidine 11 is halogenated at the 5-position(compound 12) using well known methods, for example, the halogenationmethod described in A. Gangjee et al., J. Med. Chem. (2003) 46, 591.Intermediate compound 12 may be isolated and purified using standardtechniques such as chromatography, precipitation, crystallization,filtration, and the like. Alternatively, compound 12 may be isolated andused in the next step without further purification.

5-(Substituted alkynyl)-4-chloropyrrolo[2,3-d]pyrimidine 13 is preparedusing Sonigashira conditions as described in A. Gangjee et al., (J. Med.Chem. (2003) 46, 591) to yield compound 13. Intermediate compound 13 maybe isolated and purified using standard techniques such aschromatography, precipitation, crystallization, filtration, and thelike. Alternatively compound 13 may be isolated and used in the nextstep without further purification.

Compound 13 is coupled to protected 2-methyl substituted sugar 8 (thesynthesis of which is described above and by Carroll, et al.,^(11,12))using conditions well known in the art. For example,1-O-methyl-3,5-bis-O-(2,4-dichlorophenylmethyl)-2′-C-methyl-β-D-ribofuranoside8 is dissolved in a dry inert solvent, such as dichloromethane,chloroform, carbon tetrachloride and the like, and then the solution iscooled to about 0° C. Afterwards, an excess of HBr or other appropriatereagent, in acetic acid, is added drop wise. This reaction is run fortypically at about 0° C. for about 1 hour or at ambient temperature forabout 2.5 hours or until substantially complete as determined byconventional techniques such as tlc. The resulting brominated sugarmixture is isolated and purified using standard techniques such aschromatography, precipitation, crystallization, filtration, and thelike. Alternatively this intermediate may be isolated and used in thenext step without further purification. The resulting brominated sugarmixture is co-evaporated, preferably with dry toluene, dissolved in asuitable inert diluent such as dry acetonitrile and stirred with thesodium salt of compound 13 at room temperature over night. The sodiumsalt of compound 13 is prepared in an inert atmosphere by suspendingcompound 13 in a dry inert solvent such as, acetonitrile and the like,with NaH dispersed in oil. The reaction is run for about 2 to about 24hours at a temperature of about 0 to about 40° C. Finally compound 15 isisolated and purified using standard techniques such as chromatography,precipitation, crystallization, filtration, and the like. Alternatively,this intermediate may be isolated and used in the next step withoutfurther purification.

Deprotection of compound 15 using standard methods affords compound 16,which is, in some cases, converted to the 4-amino derivative (compound17) using methods well known in the art. For example, compound 16 isadded to liquid ammonia at about −80° C. and is warmed to about 80° C.for about 24 to about 48 hours. Compound 17 is isolated and purifiedusing standard techniques such as chromatography, precipitation,crystallization, filtration, and the like.

In an alternative approach, the 1-[5-(substitutedalkynyl)-4-aminopyrrolo[2,3-d]pyrimidine]-2′-C-methyl-β-D-ribofuranosidecompounds can be prepared using the method illustrated in Scheme 4below.

Compound 12, prepared as described above is coupled to the protectedsugar 8, using the techniques described for the preparation of compound15 in Scheme 3 above to provide for compound 19. Deprotection ofcompound 19, using standard methods, provides compound 20.

From compound 20, the 1-[5-(substitutedalkynyl)-4-amino-pyrrolo[2,3-d]pyrimidine]-2′-C-methyl-β-D-ribofuranosidecompounds can be prepared by first converting the 4-chloro group to anamino group, using the technique described for the preparation ofcompound 17 from compound 16 in scheme 3 above to provide for compound21, followed by coupling using Sonigashira conditions as described in A.Gangjee et al., (J. Med. Chem. (2003) 46, 591) to form the substitutedalkynyl derivative, compound 17.

In some cases the 1-[5-(substitutedalkynyl)-4-amino-pyrrolo[2,3-d]pyrimidine]-2′-C-methyl-β-D-ribofuranosidecompounds can be prepared from compound 20 by first converting the5-iodo group to the the 5-substituted alkynyl derivative (compound 16),followed by conversion of the 4-chloro group to the amine (compound 17).The methods for each of these transformations are detailed above.

Preparation of compounds where W, W¹ or W² is other than hydrogen, usingthe compounds prepared in Schemes 3 and 4 above as the startingmaterials, can be accomplished using the methods described in thefollowing reviews of prodrug preparation:

-   1) Cooperwood, J. S. et al., “Nucleoside and Nucleotide prodrugs,”    in Ed(s) Chu, C. K. Recent Advances in Nucleosides (2002), 92-147.-   2) Zemlicka, J. et al., Biochimica et Biophysica Acta (2002),    158(2-3), 276-286.-   3) Wagner, C. et al., Medicinal Research Reviews (2002), 20(6),    417-451.-   4) Meier, C. et al., Synlett (1998), (3), 233-242.

For example, conversion of the 5′-hydroxyl group of the1-[5-(substitutedalkynyl)-4-amino-pyrrolo[2,3-d]pyrimidine]-2′-C-methyl-β-D-ribofuranosidecompounds to a phospho, diphospho or triphospho-analog can preparedusing the methods describe in D. W. Hutchinson, (Ed. Leroy b. Townsend)“The Synthesis, reaction and Properties of Nucleoside Mono-, Di-, Tri-,and tertaphosphate and Nucleosides with Changes in the PhosphorylResidue,” Chemistry of Nucleosides and Nucleotides, Plenum Press, (1991)2.

The preparation of amino acid esters on the ribofuranoside can beaccomplished as shown in Scheme 5 below:

The desired Boc-protected amino acid and N,N′-carbonyldiimidazole aredissolved in an inert solvent such as THF. The reaction mixture is heldbetween about 20 and about 40° C. for about 0.5 to 24 hours. A solutioncontaining an slight excess of the desired nucleoside in an inertsolvent such as DMF, is added to the Boc-protected amino acid mixtureand is heated at about 40 to about 80° C. for about 2 to about 24 hours.A mixture of structural isomers is isolated and separated usingconventional techniques such as evaporation, precipitation, filtration,crystallization, chromatography and the like.

The desired ester is then acidified using, for example, 1:1 v/v TFA/DCMsolution for about 0. 1 to about 1 hour about 20 and about 40° C. andevaporated. The residue is dissolved in water and held at about 0 toabout 30° C. for about 2 to about 24 hours. The mixture can be separatedand the desired product isolated by RP-HPLC using standard techniquesand conditions.

While the scheme above demonstrates the production of deazapurineprodrugs, this process can be used on any desired nucleoside compound.Likewise, the amino acid may be protected with any protective groupappropriate to the reaction conditions. These protective groups are wellknown in the art.

where T is as defined above.

Compound 1 is dissolved in a dry solvent, such as pyridine, and asilylhalide, such as tert-butylchlorodiphenylsilane, is added to form aprotecting group at the 5′-position on the sugar. Any protecting groupwhich can be directed to the 5′-position and can be removed orthongallyto the final desired 3′-ester can be used. This reaction is run forabout 4 to 24 hours at a temperature of about 10 to 40° C. The desiredacyl chloride is added to the protected nucleoside, compound 30, andstirred for about 4 to about 24 hours to form compound 31. Which can beisolated and purified using standard techniques such as isolation,crystallization, extraction, filtration, chromatography and the like.Compound 32 is prepared by removing the protecting group at the5′-position. This can be accomplished by reacting Compound 30 with a 1Msolution of tetrabutylammonium fluoride in THF. The final product isisolated and purified using standard techniques such as isolation,crystallization, extraction, filtration, chromatography and the like.

While the scheme above demonstrates the production of deazapurineprodrugs, this process can be used on any desired nucleoside compound

Utility, Testing, and Administration

Utility

The present invention provides novel compounds possessing antiviralactivity, including hepatitis C virus. The compounds of this inventioninhibit viral replication by inhibiting the enzymes involved inreplication, including RNA dependent RNA polymerase. They may alsoinhibit other enzymes utilized in the activity or proliferation ofviruses in the flaviviridae family, such as HCV.

The compounds of the present invention can also be used as prodrugnucleosides. As such they are taken up into the cells and can beintracellularly phosphorylated by kinases to the triphosphate and arethen inhibitors of the polymerase (NS5b) and/or act aschain-terminators.

Compounds of this invention may be used alone or in combination withother compounds to treat viruses.

Administration and Pharmaceutical Composition

In general, the compounds of this invention will be administered in atherapeutically effective amount by any of the accepted modes ofadministration for agents that serve similar utilities. The actualamount of the compound of this invention, i.e., the active ingredient,will depend upon numerous factors such as the severity of the disease tobe treated, the age and relative health of the subject, the potency ofthe compound used, the route and form of administration, and otherfactors. The drug can be administered more than once a day, preferablyonce or twice a day.

Therapeutically effective amounts of compounds of Formula I may rangefrom approximately 0.05 to 50 mg per kilogram body weight of therecipient per day; preferably about 0.01-25 mg/kg/day, more preferablyfrom about 0.5 to 10 mg/kg/day. Thus, for administration to a 70 kgperson, the dosage range would most preferably be about 35-70 mg perday.

In general, compounds of this invention will be administered aspharmaceutical compositions by any one of the following routes: oral,systemic (e.g., transdermal, intranasal or by suppository), orparenteral (e.g., intramuscular, intravenous or subcutaneous)administration. The preferred manner of administration is oral using aconvenient daily dosage regimen that can be adjusted according to thedegree of affliction. Compositions can take the form of tablets, pills,capsules, semisolids, powders, sustained release formulations,solutions, suspensions, elixirs, aerosols, or any other appropriatecompositions. Another preferred manner for administering compounds ofthis invention is inhalation. This is an effective method for deliveringa therapeutic agent directly to the respiratory tract (see U.S. Pat. No.5,607,915).

The choice of formulation depends on various factors such as the mode ofdrug administration and bioavailability of the drug substance. Fordelivery via inhalation the compound can be formulated as liquidsolution, suspensions, aerosol propellants or dry powder and loaded intoa suitable dispenser for administration. There are several types ofpharmaceutical inhalation devices-nebulizer inhalers, metered doseinhalers (MDI) and dry powder inhalers (DPI). Nebulizer devices producea stream of high velocity air that causes the therapeutic agents (whichare formulated in a liquid form) to spray as a mist that is carried intothe patient's respiratory tract. MDI's typically are formulationpackaged with a compressed gas. Upon actuation, the device discharges ameasured amount of therapeutic agent by compressed gas, thus affording areliable method of administering a set amount of agent. DPI dispensestherapeutic agents in the form of a free flowing powder that can bedispersed in the patient's inspiratory air-stream during breathing bythe device. In order to achieve a free flowing powder, the therapeuticagent is formulated with an excipient such as lactose. A measured amountof the therapeutic agent is stored in a capsule form and is dispensedwith each actuation.

Recently, pharmaceutical formulations have been developed especially fordrugs that show poor bioavailability based upon the principle thatbioavailability can be increased by increasing the surface area i.e.,decreasing particle size. For example, U.S. Pat. No. 4,107,288 describesa pharmaceutical formulation having particles in the size range from 10to 1,000 nm in which the active material is supported on a crosslinkedmatrix of macromolecules. U.S. Pat. No. 5,145,684 describes theproduction of a pharmaceutical formulation in which the drug substanceis pulverized to nanoparticles (average particle size of 400 nm) in thepresence of a surface modifier and then dispersed in a liquid medium togive a pharmaceutical formulation that exhibits remarkably highbioavailability.

The compositions are comprised of in general, a compound of Formula I incombination with at least one pharmaceutically acceptable excipient.Acceptable excipients are non-toxic, aid administration, and do notadversely affect the therapeutic benefit of the compound of Formula T.Such excipient may be any solid, liquid, semi-solid or, in the case ofan aerosol composition, gaseous excipient that is generally available toone of skill in the art.

Solid pharmaceutical excipients include starch, cellulose, talc,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, dried skim milk and the like. Liquid and semisolid excipientsmay be selected from glycerol, propylene glycol, water, ethanol andvarious oils, including those of petroleum, animal, vegetable orsynthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesameoil, etc. Preferred liquid carriers, particularly for injectablesolutions, include water, saline, aqueous dextrose, and glycols.

Compressed gases may be used to disperse a compound of this invention inaerosol form. Inert gases suitable for this purpose are nitrogen, carbondioxide, etc. Other suitable pharmaceutical excipients and theirformulations are described in Remington's Pharmaceutical Sciences,edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990).

The amount of the compound in a formulation can vary within the fullrange employed by those skilled in the art. Typically, the formulationwill contain, on a weight percent (wt %) basis, from about 0.01-99.99 wt% of a compound of Formula I based on the total formulation, with thebalance being one or more suitable pharmaceutical excipients.Preferably, the compound is present at a level of about 1-80 wt %.Representative pharmaceutical formulations containing a compound ofFormula I are described below.

Additionally, the present invention is directed to a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof the present invention in combination with a therapeutically effectiveamount of another active agent against RNA-dependent RNA virus and, inparticular, against HCV. Agents active against HCV include, but are notlimited to, ribavirin, levovirin, viramidine, thymosin alpha-1, aninhibitor of HCV NS3 serine protease, interferon-α, pegylatedinterferon-α (peginterferon-α), a combination of interferon-α andribavirin, a combination of peginterferon-α and ribavirin, a combinationof interferon-α and levovirin, and a combination of peginterferon-α andlevovirin. Interferon-α includes, but is not limited to, recombinantinterferon-α2a (such as Roferon interferon available fromHoffman-LaRoche, Nutley, N.J.), interferon-α2b (such as Intron-Ainterferon available from Schering Corp., Kenilworth, N.J., USA), aconsensus interferon, and a purified interferon-α product. For adiscussion of ribavirin and its activity against HCV, see J. O. Saundersand S. A. Raybuck, “Inosine Monophosphate Dehydrogenase: Considerationof Structure, Kinetics and Therapeutic Potential,” Ann. Rep. Med. Chem.,35:201-210 (2000).

EXAMPLES

The examples below as well as throughout the application, the followingabbreviations have the following meanings. If not defined, the termshave their generally accepted meanings.

-   AcOH or HOAc=acetic acid-   Ac₂O=acetic anhydride-   Ar=aryl hydrogen-   atm=atmosphere-   Boc=t-butoxycarbonyl-   bs=broad singlet-   CAN=ceric ammonium nitrate-   cm=centimeter-   d=doublet-   con=concentration-   DCM=dichloromethane-   dd=doublet of doublets-   DMF=Dimethylformamide-   dt=doublet of triplets-   DBU=1,8-diazabicyclo[5.4.0]undec-7-ene-   DCB=2,4-dichlorobenzyl-   DCC=dicyclohexylcarbodiimide-   DMEM=Delbecco's minimum eagles medium-   DMSO=Dimethylsulfoxide-   DTT=Dithiothreitol-   EDTA=ethylene diamine tetraacetic acid-   eq. or eq=Equivalents-   g=Gram-   h or hr=Hour-   HCV=hepatitis C virus-   HPLC=high performance liquid chromatography-   IPTG=Isopropyl β-D-1-thiogalactopyranoside-   IU=international units-   kb=Kilobase-   kg=Kilogram-   KOAc=potassium acetate-   L=Liters-   M=Multiplet-   M=Molar-   Me=Methyl-   MeOH=Methanol-   min=Minute-   mg=Milligram-   mL=Milliliter-   mm=Millimeters-   mM=millimolar-   mmol=millimol-   MS=mass spectrum-   ng=nanograms-   N=Normal-   nm=nanometers-   nM=nanomolar-   NMR=nuclear magnetic resonance-   NTA=nitrilotriacetic acid-   NTP=nucleotide triphosphate-   RP HPLC=reverse phase high performance liquid chromatography-   q=Quartet-   s=Singlet-   t=Triplet-   TBAF=Tetrabutyl ammonium fluoride-   TEA=Triethylamine-   TFA=trifluoroacetic acid-   THF=Tetrahydrofuran-   tlc or TLC=thin layer chromatography-   T_(m)=Melting temperature-   UTP=uridine triphosphate-   μL=microliters-   μg=micrograms-   μM=micromolar-   v/v=volume to volume-   w/w=weight to weight-   Wt %=weight percent

In addition, all reaction and melting temperatures are in degreesCelsius unless reported otherwise.

In the examples below as well as elsewhere throughout this application,the claimed compounds employ the following numbering system:

Example 1 Preparation of the intermediate1-O-methyl-2-methyl-3,5-bis-O-(2,4-dichlorobenzyl)-β-D-ribofuranose

Step 1: Preparation of1-O-methyl-2,3,5-tris-O-(2,4-dichlorobenzyl)-β-D-ribofuranose

The title compound is synthesized using the methods described in Marin,P.; Helv. Chim. Acta, 1995, 78, 486 starting with commercially availableD-ribose.

Step 2: Preparation of1-O-methyl-3,5-bis-O-(2,4-dichlorobenzyl)-β-D-ribofuranose

To a solution of the product of Step 1 (171.60 g, 0.2676 mol) in 1.8 Lof methylene chloride that was cooled to 0° C., was added dropwise asolution of stannous chloride (31.522 mL, 0.2676 mol) in 134 mL ofmethylene chloride while stirring. After maintaining the solution atabout 3° C. for approximately 27 hours, another 5.031 mL of stannouschloride (SnCl₄) (0.04282 mol) was added and the solution was kept atabout 3° C. overnight. After a total reaction time of approximately 43hours, the reaction was quenched by carefully adding the solution to 1.9L of a saturated NaHCO₃ solution. Tin salts were removed via filtrationthrough Celite after which the organic phase was isolated, dried withMgSO₄ and evaporated in vacuo. The yield of raw, dark yellow oil was173.6 g. The crude oil was used directly in the next step withoutfurther purification.

Step 3: Preparation of1-O-methyl-2-oxo-3,5-bis-O-(2,4-dichlorobenzyl)-β-D-ribofuranose

To an ice cold solution of Dess-Martin periodinane (106.75 g, 0.2517mol) in 740 mL anhydrous methylene chloride, under argon, was added asolution of the product of Step 2 above in 662 mL anhydrous methylenechloride over 0.5 hours. The reaction mixture was stirred at 0° C. for0.5 hours and then at room temperature for 6 days. The mixture wasdiluted with 1.26 L of anhydrous diethyl ether and then poured into anice-cold mixture of Na₂S₃O₃.5H₂O (241.2 g, 1.5258 mol) in 4.7 L ofsaturated aqueous sodium bicarbonate. The layers were separated, and theorganic layer was washed with 1.3 L of saturated aqueous sodiumbicarbonate, 1.7 L water and 1.3 L brine, dried with MgSO₄, filtered andevaporated to give the target compound. The compound (72.38 g, 0.1507mol) was used without further purification in the next step.

Step 4: Preparation of the title compound

A solution of MeMgBr in 500 mL anhydrous diethyl ether maintained at−55° C. was added dropwise to a solution of the product of step 3 above(72.38 g, 0.1507 mol) also in 502 mL of anhydrous diethyl ether. Thereaction mixture was allowed to warm to −30° C. and stirred mechanicallyfor 4 hours at about −30° C. to −15° C., then poured into 2 L ice coldwater. After stirring vigorously at ambient temperature for 0.5 hours,the mixture was filtered through a Celite pad (14×5 cm), which wasthoroughly washed with diethyl ether. The organic layer was dried withMgSO₄, filtered and concentrated in vacuo. The residue was dissolved inhexanes (˜1 mL per gram crude), applied to a silica gel column (1.5 Lsilica gel in hexanes) and eluted with hexanes and 4:1 hexanes:ethylacetate (v/v) to give 53.58 g (0.1080 mol) of the final purifiedproduct. The morphology of the title compound was that of an off-yellow,viscous oil;

-   MS: m/z 514.06 (M+NH₄+).

Example 2 Preparation of7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-[2-(trimethylsilylethyn-1-yl]-pyrrolo[2,3-d]pyrimidine

Step 1. 4-Chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine:

4-Chloro-7H-pyrrolo[2,3-d]pyrimidine 10.75 g (70 mmol) andN-iodosuccinimide (16.8 g, 75 mmol) were dissolved in 400 mL of dry DMFand left at ambient temperature in the darkness over night. The solventwas evaporated. The yiellow residue was suspended in hot 10% solution ofNa₂SO₃, filtered, washed twice with hot water and crystallized fromethanol to yield 14.6 g (74.6%) of the title compound as off-whitecrystals. The mother liquid was evaporated up to ⅓ volume andcrystallized again from ethanol to give 2.47 g (12.3%) of the titleproduct. The total yield is close to 100%;

T_(m) 212-214° C. (dec); UV λ_(max): 307, 266, 230, 227 nm (methanol);MS: 277.93 (M−H), 313 (M+Cl); ¹H-NMR (DMSO-d6): 12.94 (s, 1H, NH), 8.58(s, 1H, H-2), 7.94 (s, 1H, H-8).

Step 2.7-(2′-methyl-3′,5′-bis-O-(2,4-dichlorobenzyl)-β-D-ribofuranosyl)-4-chloro-5-iodo-pyrrolo[2,3-d]pyrimidine:

The base, obtained as described above (11.2 g, 40 mmol) was suspended in500 mL of CH₃CN, NaH was added (1.6 g, 40 mmol 60% in oil) and thereaction mixture was stirred at room temperature until NaH was dissolved(about 2 hour). 1-O-Methyl-2-methyl-3,5-bis-O-(2,4-dichlorobenzyl)-β-D-ribofuranose (10 g, 20mmol) was dissolved in 500 mL of DCM and cooled down to 4° C. inice/water bath. HBr_((g)) was bubbled through the solution for about 30min. The reaction was monitored by TLC and run until the disappearanceof the starting sugar (ether/hexane 1:9 v/v). Upon reaction completion,the solvent was evaporated at the temperature not higher that 20° C. andkept for 20 min in deep vacuum to remove the traces of HBr. Solution ofNa-salt of the base was fast filtrated and the filtrate was added to thesugar component. The reaction was kept overnight at ambient temperature,neutralized with 0.1 N H₂SO₄ and evaporated. The residue was distributedbetween 700 mL of ethyl acetate and 700 mL of water. Organic fractionwas washed with water (150 mL), brine (150 mL), dried over Na₂SO₄ andevaporated to give semi crystalline mixture. Toluene (500 mL) was addedto form light tan precipitate of nonreacted heterocyclic base 2.5 g(25%). Filtrate was concentrated up to the volume of 50 mL and loaded onthe glass filter with silica gel (10×10 cm). The filter was washed with10% ethyl acetate in toluene collecting 500 mL fractions. Fraction 2-4contained the title compound; fractions 6-7 contained the heterocyclicbase.

Fractions 2-4 were evaporated, ether was added to the colorless oil andthe mixture was sonicated for 5 min. The off-white precipitate wasformed, yield 7.4 g (50%), mother liquid was evaporated and thedescribed procedure was repeated to yield 0.7 g more of the titlenucleoside. Total yield is 8.1 g (54.4%);

T_(m): 67-70° C.; ¹H-NMR (DMSO-d₆): δ 8.66 (s, 1H), 8.07 (s, 1H),7.62-7.34 (m, 6H), 6.22 (s, 1H), 5.64 (s, 1H), 4.78-4.55 (m, 4H), 4.20(s, 2H), 3.97-3.93 and 3.78-3.75 (dd, 1H), 0.92 (s, 3H); MS: 743.99(M+H); Recovered base (total): 4 g as off-white crystals; T_(m) 228-230°C.

Step 3.7-(2′-methyl-β-D-ribofuranosyl)-4-chloro-5-iodo-pyrrolo[2,3-d]pyrimidine:

To the solution of the compound from the previous step (8 g, 10.7 mmol)in DCM (200 mL) at −78° C. was added boron trichloride (1M in DCM, 88mL, 88 mmol) dropwise. The mixture was stirred at -78° C. for 2.5 hoursand additionally overnight at −20° C. The reaction was quenched byaddition of MeOH/DCM (90 mL, 1:1) and the resulting mixture stirred at−20° C. for 30 min, then neutralized by aqueous ammonia at the sametemperature. The solid was filtered and washed with methanol/DCM (250mL, 1:1). The filtrates were combined with 50 mL of silica gel andevaporated up to dryness. Dry silica was loaded on the glass filter withsilica gel (10×10 cm). The filter was washed with ethyl acetatecollecting 500 mL fractions. Fraction 2-4 contained the title compound.The solvent was evaporated and the residue crystallized fromacetone/hexane to give 3.3 g (72%) of title nucleoside;

¹H-NMR (DMSO-d₆): δ 8.84 (s, 1H), 8.20 (s, 1H), 6.21 (s, 1H), 4.00-3.60(m, sugar), 0.84 (s, 3H); MS: 426.26 (M+H); T_(m):182-185° C.

Step 4.7-(2′-methyl-β-D-ribofuranosyl)-4-amino-5-iodo-pyrrolo[2,3-d]pyrimidine:

Nucleoside (1.5 g, 3.5 mmol) prepared above was treated with liquidammonia at 85° C. for 24 hours in the metal pressure reactor. Afterevaporation of ammonia the residue was dissolved in methanol andco-evaporated with silica gel (about 20 mL). Silica gel bearing theproduct was on the column (5×10 cm) with silica gel in acetonecollecting 50 mL fractions. Fractions 2-8 contained the titled compound.Acetone was evaporated and the residue crystallized frommethanol/acetonitrile to give 1.2 g (84%) of the titled nucleoside;

T_(m)220-222° C. (dec); ¹H-NMR (DMSO-d₆): δ 8.20 (s, 1H), 7.80 (s, 1H),6.80-6.50 (bs, 1H), 6.09 (s, 1H), 5.19 (t, 1H, sugar), 5.13-5.11 (m, 2H,sugar), 4.00-3.70 (m, 3H, sugar), 3.60-3.20 (m, 1H, sugar), 0.84 (s,3H); MS 407.32 (M+H).

Step 5.7-(2′-methyl-β-D-ribofuranosyl)-4-amino-5-(trimethylsilanylethyn-1-yl)-pyrrolo[2,3-d]pyrimidine:

Aminonucleoside synthesized in previous step (1.7 g, 4.2 mmol) wasdissolved in the mixture of 12 mL of dry DMF and 28 mL of dry THF.Triethylamine (3.6 mmol, 0.5 mL), CuI (1 mmol, 80 mg) were added and theflask was filled with argon. Tetrakis(triphenylphosphine)palladium(0)(0.04 mmol, 46 mg) followed with (trimethylsilyl)acetylene was added andthe mixture was stirred under argon for 20 hours.

The solvent was evaporated and the residue in acetone was filteredthrough a silica gel (5×10 cm). The acetone was evaporated, the residuewas dissolved in acetonitrile and then filtered again through the silicagel column of the same size; an elution was made with pure acetonitrile.The acetonitrile was concentrated up to small volume, about 10 volumesof ether were added and the solution was sonicated for 5 min. Whitecrystals of the titled compound were formed, yield 0.8 g (71%);

T_(m) 188-191° C. (decompose); ¹H-NMR (DMSO-d₆): δ 8.17 (s, 1H), 7.92(s, 1H), 7.20-6.80 (t, 1.2H), 5.83 (s, 1H), 3.75-3.20 (m, sugar), 0.45(s, 3H), 0 (s, 9H).

Example 3 Preparation of7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-[2-(pyrid-2-yl)ethyn-1yl]-pyrrolo[2,3-d]pyrimidine

To a solution of the compound from Step 4, Example 2 in DMF (0.05M) isadded 1.0 equivalent TEA, 0.4 eq CuI, 6.0 equivalents2-ethynyl-pyridine. This mixture is degassed with argon and 10 mole % ofP(Ph₃)₄Pd is added and the reaction stirred for 24 hours between 25-80°C. The reaction mixture is then concentrated in vacuo, taken up in DMFand purified by RP HPLC on PHenominex column (250×20 mm) using gradientof acetonitrile in water from 0 to 80% over 30 min at 10 mL/min.

Example 4 Preparation of7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-(2-carboxamidoethyn-1yl)-pyrrolo[2,3-d]pyrimidine

To a solution of the product from Example 8 (20 mg, 0.053 mmol) wasadded 1.0 mL concentrated ammonia solution (30% aqueous solution) andstirred at room temp for 1 hour. The resulting precipitate was filteredand dried via co-evaporation with ethanol to yield 15 mg (80%) of thetitle compound;

¹H-NMR (DMSO-d₆): δ 8.26 (bs, 1H), 8.16 (s, 1H), 8.14 (s, 1H), 7.56 (bs,1H), 7.2-6.4(bs, 2H), 6.11 (s, 1H), 5.26-5.14 (m, 3H), 3.9-3.6 (m, 4H,sugar), 0.69 (s, 3H); MS 348.10 (M+H).

Example 5 Preparation of7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-[3,3-diethoxyproparg-1-yl)-pyrrolo[2,3-d]pyrimidine

To a solution of compound from Step 4, Example 2 (100 mg, 0.246 mmol) in6.5 mL THF-DMF (3:1 v/v) was added CuI (18.2 mg, 0.096 mmol), TEA (32μL, 0.23 mmol), propiolaldehyde diethyl acetal (0.05 mL, 0.36 mmol). Themixture was degassed with argon and P(Ph₃)₄Pd (28 mg, 0.024 mmol) wasadded and the reaction was stirred at room temperature for 3.5 hours. Asecond charge of propiolaldehyde diethyl acetal (0.05 mL) was added andthe reaction was allowed to stir at room temperature overnight. Thereaction was then concentrated in vacuo and purified on silica gel(plated on gel with 100% CH₂Cl₂, eluded with 15% MeOH-CH₂Cl₂) to yield60 mg (60%) of the title compound;

¹H-NMR (CD₃OD): δ 8.11 (s, 1H), 7.90 (s, 1H), 6.22 (s, 1H), 4.2-3.6 (m,4H, sugar), 3.8-3.6 (m, 4H), 1.26 (t, 6H), 0.84 (s, 3H); MS 407.22(M+H).

Example 6 Preparation of7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-[2-(4-methoxyphenyl)ethyn-1yl]-pyrrolo[2,3-d]pyrimidine

To a solution of the compound from Step 4, Example 2 in DMF is added 1.0equivalent TEA, 0.4 eq CuI, 6.0 equivalents 1-ethynyl-4-methoxy-benzene.This mixture is degassed with argon and 10 mole % of P(Ph₃)₄Pd is addedand the reaction stirred for 24 hours between 25-80° C. The reactionmixture is then concentrated in vacuo, taken up in DMF and purified byRP HPLC on PHenominex column (250×20 mm) using gradient of acetonitrilein water from 0 to 80% over 30min at 10 mL/min.

Example 7 Preparation of7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-(2-phenylethyn-1yl)-pyrrolo[2,3-d]pyrimidine

A solution was made of the product from Step 4, Example 2 (50.0 mg, 0.1231 mmol) in 5 mL dimethylformamide. The solution was degassed bybubbling with argon while sonicating for 5 min. To this solution wasadded triethylamine (16.0 μL, 0.1145 mmol), copper iodide (9.4 mg,0.0492 mmol), and tetrakis(triphenylphosphine) palladium (0) (14.2 mg,0.0123 mmol). Next, phenylacetylene (81.1 μL, 0.7386 mmol) was added.The mixture was stirred under argon for 4 hours. Upon completion, themixture was concentrated in vacuo. The reaction crude was dissolved in 1mL dimethylformamide, diluted to 50 mL with deionized water and thenwashed through a celite pad. The solution was again concentrated down todryness, then redissolved in 1.0 mL dimethylformamide and 3.5 mL water.The title compound was purified by HPLC;

¹H-NMR (CD₃OD): 8.14 (s, 1H), 7.91 (s, 1H), 7.54-7.51 (m, 2H), 7.40-7.36(m, 3H), 6.25 (s, 1H), 4.16-3.85 (m, 4H), 0.87 (s, 3H). MS: 381.14(m/z).

Example 8 Preparation of7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-(ethyl2-carboxylethyn-1yl)-pyrrolo[2,3-d]pyrimidine

To a solution of the product from Step 4, Example 2 (450.0 mg, 1.108mmol) in 28.8 mL THF-DMF (2:1 v/v) was added CuI (0.082 g, 0.432 mmol),TEA (144 μL, 1.035 mmol), tetrakis(triphenylphosphine)palladium(0)(0.126 g, 0.108 mmol) and the solution degassed with argon. Ethylpropiolate (100 μL, 1.0 11 mmol) was added and the reaction was heatedto 55° C. An additional 100 μL of ethyl propiolate was added every hourfor six hours until no starting material of Example 2, Step 4, waspresent by TLC. The reaction mixture was concentrated in vacuo, taken upin DMF and purified by RP HPLC on PHenominex column (250×20 mm) usinggradient of acetonitrile in water from 0 to 60% over 30 min at 10 mL/minto yield 115 mg (28%) of the title compound;

¹H-NMR (CD₃OD): 8.24 (s, 1H), 8.17 (s, 1H), 6.24 (s, 1H), 4.28 (q, 2H),4.13-3.87 (m, 4H, sugar), 1.34(t, 3H), 0.86 (s, 3H); MS 377.11 (M+H).

Example 9 Preparation of7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-(2-carboxylethyn-1yl)-pyrrolo[2,3-d]pyrimidine

To the product from Example 8 (20.0 mg, 0.053 mmol) was added 500 μL of1N NaOH and stirred at room temperature for 30 min. The reaction wasthen diluted with water and purified by RP HPLC on Phenominex column(250×20 mm) using gradient of acetonitrile in water from 0 to 60% over30 min at 10 mL/min to yield 7.0 mg (38%) of the title compound;

¹H-NMR (CD₃OD): 8.10 (s, 1H), 7.96 (s, 1H), 6.22 (s, 1H), 4.12-3.82 (m,4H, sugar), 0.84 (s, 3H); MS: 349.10 (M+H).

Example 10 Preparation of7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-(2-cis-methoxyethen-1-yl)-pyrrolo[2,3-d]pyrimidine

Step 1. Preparation of7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-(formyl)-pyrrolo[2,3-d]pyrimidine:

7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-(formyl)-pyrrolo[2,3-d]pyrimidinewas synthesized according to the procedure described by Shin-ichiWatanabe and Tohru Ueda in Nucleosides and Nucleotides (1983) 2(2),113-125. In the synthesis of the title compound, however,7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-pyrrolo[2,3-d]pyrimidine (SeeCarroll, et al.,^(11,12)) was substituted in place of tubercidin;

H¹-NMR (CD₃OD): 0.9 (s, 3H, 2′-CH₃), 3.8-4.2 (m, 4H, sugar), 6.3 (s, 1H,1′-H), 8.2 and 8.6 (s, 1H, —Ar), 9.7 (s, 1H, -aldehyde-H); MS: 309.13(M+H).

Step 2. Preparation of the Title Compound:

The product from Step 1 above (0.050 g, 0.162 mmol) was dissolved in 2mL DMSO and added dropwise to the preformed ylide of(methoxymethyl)-triphenylphosphonium chloride and stirred at roomtemperature. The ylide was formed by dissolving (0.555 g, 1.62 mmol)(methoxymethyl)triphenylphosphonium chloride in 10 mL DMSO and adding0.81 mL 2 M methylsulfinyl carbanion solution (1.62 mmol) and stirred atroom temperature for 15 min. After stirring at room temperature for 4hours, a second charge of (1.62 mmol) of the ylide of(methoxy-methyl)triphenyl-phosphonium chloride was added to the reactionand the reaction was allowed to stir at room temperature overnight. Thereaction was quenched with water and diluted with methylene chloride.The layers were separated and the organic layer was extracted with watertwice. The aqueous layers were combined and concentrated in vacuo andpurified by RP HPLC on Phenominex column (250×20 mm) using gradient ofacetonitrile in water from 0 to 30% over 30 min at 10 mL/min to yield 20mg of the trans enol ether and 10 mg of cis isomer, (56%) yield;

(cis-isomer) H¹-NMR (CD₃OD): 0.82 (s, 3H, 2′-CH₃), 3.8 (s, 3H, —OCH₃)3.8-4.2 (m, 4H, sugar), 5.56 (d, 1H), 6.23 (s, 1H, 1′-H), 6.25 (d, 1H),7.8 and 8.0 (s, 1H, —Ar).

Example 11 Preparation of7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-(ethen-1-yl)-pyrrolo[2,3-d]pyrimidine

The title compound from Example 2 (0.040 g, 0.0132 mmol) was dissolvedin methanol and NH₄OH was added and the mixture left at ambienttemperature for 1 hour. The solvent was evaporated, the residuedissolved in methanol and co-evaporated with silica gel. Dry silica wasloaded on the glass filter with silica gel and the desilylated compoundwas eluted with acetone. Solvent was evaporated and the residuecrystallized from methanol/acetonitrile to provide for7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-(ethyn-1-yl)-pyrrolo[2,3-d]pyrimidine.

T_(M) 209-219° C. (decomposition); MS 305.13 (M+H); ¹H-NMR (DMSO-d6):8.10 (s, 1H, H-2), 7.94 (s, 1H, H-8), 6.08 (s, 1H, H-1′), 5.32-5.13 (m,3H, sugar), 3.96-3.62 (m, 4H, sugar), 0.68 (s, 3H, methyl).

The acetylene product prepared above was dissolved in 3 mL THF and 22 mgof Lindlar's catalyst was added. The solution was stirred at ambienttemperature under 1 atm of hydrogen (via balloon) for 7 days. Theballoon was recharged with hydrogen at the beginning of each day. After7 days, the reaction was filtered through celite to remove catalyst,concentrated in vacuo, and purified by reverse phase HPLC on Phenominexcolumn (250×20 mm) using a gradient of acetonitrile in water from 0 to30% over 30 min at 10 mL/min to yield 30 mg (75% yield) of the titlecompound;

H¹-NMR (CD₃OD): 0.835 (s, 3H, 2′-CH₃), 3.8-4.2 (m, 4H, sugar), 5.2 (dd,1H), 5.5 (dd, 1H), 6.23 (s, 1H, 1′-H), 6.9 ((dd, 1H), 7.73 and 8.06 (s,1H, —Ar); MS: 307.15 (M+H).

Example 12 Preparation of7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-(formyl)-pyrrolo[2,3-d]pyrimidine

Step 1. 4-Chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine:

4-Chloro-7H-pyrrolo[2,3-d]pyrimidine 10.75 g (70 mmol) andN-iodosuccinimide (16.8 g, 75 mmol) were dissolved in 400 mL of dry DMFand left at ambient temperature in the darkness over night. The solventwas evaporated. The yellow residue was suspended in hot 10% solution ofNa₂SO₃, filtered, washed twice with hot water and crystallized fromethanol to yield 14.6 g (74.6%) of the title compound as off-whitecrystals. The mother liquid was evaporated up to ⅓ volume andcrystallize again from ethanol to give 2.47 g (12.3%) of the titleproduct;

Total yield is close to 100%; T_(m): 212-214 (decompose); UV λ_(max):307, 266, 230, 227 nm (methanol); MS: 277.93 (M−H), 313 (M+Cl); ¹H-NMR(DMSO-d₆): δ 12.94 (s, 1H), 8.58 (s, 1H), 7.94 (s, 1H).

Step 2.7-(2′-methyl-3′,5′-bis-O-(2,4-dichlorobenzyl)-β-D-ribofuranosyl)-4-chloro-5-iodo-pyrrolo[2,3-d]pyrimidine:

The base, obtained as described above (11.2 g, 40 mmol) was suspended in500 mL of CH₃CN, NaH was added (1.6 g, 40 mmol 60% in oil) and thereaction mixture was stirred at room temperature until NaH was dissolved(about 2 hour). 1-O-Methyl-2-methyl-3,5-bis-O-(2,4-dichlorobenzyl)-β-D-ribofuranose (10 g, 20mmol) was dissolved in 500 mL of DCM and cooled down to 4° C. inice/water bath. HBr—gas was bubbled through DCM solution about 30 min.Reaction was controlled by TLC by disappearance of the starting sugar(ether/hexane 1:9 v/v). Upon the reaction was completed the solvent wasevaporated at the temperature not higher that 20° C. and kept for 20 minin deep vacuum to remove the traces of HBr. Solution of Na-salt of thebase was fast filtrated and the filtrate was added to the sugarcomponent. The reaction was kept overnight at ambient temperature,neutralized with 0. 1 N H₂SO₄ and evaporated. The residue wasdistributed between 700 mL of ethyl acetate and 700 mL of water. Theorganic fraction was washed with water (150 mL), brine (150 mL), driedover Na₂SO₄ and evaporated to give a semi-crystalline mixture. Toluene(500 mL) was added to form a light tan precipitate of non-reactedheterocyclic base 2.5 g (25%). The filtrate was concentrated up to thevolume of 50 mL and loaded on the glass filter with silica gel (10×10cm). The filter was washed with 10% ethyl acetate in toluene collecting500 mL fractions. Fraction 2-4 contained the title compound; fractions6-7 contained the heterocyclic base.

Fractions 2-4 were evaporated, ether was added to the colorless oil andthe mixture was sonicated for 5 min. The off-white precipitate wasformed, yield 7.4 g (50%). The mother liquid was evaporated and thedescribed procedure was repeated to yield and additional 0.7 g of thetitle nucleoside. Total yield is 8.1 g (54.4%);

T_(m): 67-70° C.; ¹H-NMR (DMSO-d₆): δ 8.66 (s, 1H), 8.07 (s, 1H),7.62-7.34 (m, 6H), 6.22 (s, 1H), 5.64 (s, 1H), 4.78-4.55 (m, 4H), 4.20(s, 2H), 3.97-3.93 and 3.78-3.75 (dd, 1H), 0.92 (s, 3H); MS: 743.99(M+H); Recovered base (total): 4 g as off-white crystals; T_(m):228-230° C.

Step 3.7-(2′-methyl-β-D-ribofuranosyl)-4-chloro-5-iodo-pyrrolo[2,3-d]pyrimidine:

To the solution of the compound from the previous step (8 g, 10.7 mmol)in DCM (200 mL) at −78° C. was added boron trichloride (1 M in DCM, 88mL, 88 mmol) dropwise. The mixture was stirred at −78° C. for 2.5 hoursand additionally overnight at −20° C. The reaction was quenched byaddition of methanol/DCM (90 mL, 1:1) and the resulting mixture stirredat −20° C. for 30 min, then neutralized by aqueous ammonia at the sametemperature. The solid was filtered and washed with methanol/DCM (250mL, 1:1). The filtrates were combined with 50 mL of silica gel andevaporated up to dryness. Dry silica was loaded on the glass filter withsilica gel (10×10 cm). The filter was washed with ethyl acetatecollecting 500 mL fractions. Fraction 2-4 contained the title compound.The solvent was evaporated and the residue crystallized fromacetone/hexane to give 3.3 g (72%) of title nucleoside;

¹H-NMR (DMSO-d₆): δ 8.84 (s, 1H), 8.20 (s, 1H), 6.21 (s, 1H), 4.00-3.60(m, sugar), 0.84 (s, 3H); MS: 426.26 (M+H); T_(m): 182-185° C.

Step 4.7-(2′-methyl-β-D-ribofuranosyl)-4-amino-5-iodo-pyrrolo[2,3-d]pyrimidine:

The nucleoside (1.5 g, 3.5 mmol) prepared above was treated with liquidammonia at 85° C. for 24 hours in the metal pressure reactor. Afterevaporation of ammonia the residue was dissolved in methanol andco-evaporated with silica gel (about 20 mL). Silica gel bearing theproduct was on the column (5×10 cm) with silica gel in acetonecollecting 50 mL fractions. Fractions 2-8 contained the titled compound.Acetone was evaporated and the residue crystallized frommethanol/acetonitrile to give 1.2 g (84%) of the titled nucleoside;

T_(m) 220-222° C. (decompose); ¹H-NMR (DMSO-d₆): δ 8.20 (s, 1H), 7.80(s, 1H), 6.80-6.50 (bs, 1H), 6.09 (s, 1H), 5.19 (t, 1H), 5.13-5.11 (m,2H), 4.00-3.70 (m, 3H), 3.60-3.20 (m, 1H), 0.84 (s, 3H); MS 407.32(M+H).

Step 5: Preparation of the Title Compound:

A solution was made of the compound prepared in Step 4 above (50.0 mg,0. 1231 mmol) in 5 mL dry tetrahydrofuran, which was then purged of airby slowly bubbling with carbon monoxide. To this solution was addedtetrakis (triphenyl-phosphine)palladium(0) (2.8 mg, 0.0025 mmol). Thereaction was stirred for 10 minutes, and then heated to 50° C. Next,tributyltin hydride in THF (35.9 μL, 0.1354 mmol) was slowly added over2.5 hours—CO gas being continually bubbled through during this time.Upon completion, the mixture was concentrated in vacuo. The reactioncrude was dissolved in 1 mL dimethylformamide, diluted to 50 mL withdeionized water, and then washed through a celite pad. The solution wasagain concentrated down to dryness then redissolved in 1.0 mLdimethylformamide and 3.5 mL water. The title compound was purified byHPLC;

¹H-NMR (DMSO-d₆): δ 9.64 (s, 1H), 8.60 (s, 1H), 8.18 (s, 1H), 7.62 (m,2H), 6.14 (s, 1H), 5.28-5.19 (m, 3H), 3.94-3.71 (m, 4H), 0.75 (s, 3H);MS: 309.11 (m/z).

Example 13 Preparation of7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-(carbaldehydeoxime)-pyrrolo[2,3-d]pyrimidine

To a solution of the title compound (0.1 g, 0.325 mmol) from Example 12in 10 mL 50% ethanol was added hydroxylamine hydrochloride (0.073 g,1.05 mmol) and KOAc (0.103 g, 1.05 mmol) and heated to 60° C. for 2.5hours. The crude mixture was concentrated, diluted with water andpurified by reverse phase HPLC on Phenominex column (250×20 mm) usinggradient of acetonitrile in water from 0 to 30% over 30 min at 10 mL/minto yield 10 mg of the title compound;

¹H-NMR (CD₃OD): δ 0.86 (s, 3H), 3.8-4.2 (m, 4H), 6.21 (s, 1H), 7.78,8.06, 8.08 (s, 1H). MS: 324.15 (M+H).

Example 14 Preparation of7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-(boronicacid)-pyrrolo[2,3-d]pyrimidine

To a solution of the compound from Step 4, Example 2 (60 mg, 0.148 mmol)in 1 mL DMSO was added KOAc (44 mg, 0.449 mmol), bis(neopentylglycoloto)diboron (40 mg, 0. 177 mmol). The mixture was degassed withargon and P(Ph₃)₂PdCl₂ (3.1 mg, 0.004 mmol) was added and the reactionwas heated to 80° C. for 4 hours. The mixture was diluted with water andpurified by RP HPLC on Phenominex column (250×20 mm) using gradient ofacetonitrile in water (with from 0 to 50% over 30 min at 10 mL/min toyield 16 mg (33%) of the title compound;

¹H-NMR (D₂O ): δ 8.12 (s, 1H), 7.75 (s, 1H), 6.12 (s, 1H), 4.2-3.8 (m,4H), 0.70 (s, 3H); MS 325.13 (M+H).

Example 15 Preparation of7-(2′-C-methyl-β-D-ribofuranosyl)-4-amino-5-(diisopropoxymethyl)-pyrrolo[2,3-d]pyrimidine

To a solution of the compound from Step 5, Example 13 in anhydrousisopropanol is placed activated molecular sieves and the solution isacidified with HCl. The solution is heated to between 50-80° C. untilstarting material has been consumed. The resulting diacetal is purifiedon RP HPLC on Phenominex column (250×20 mm) using gradient ofacetonitrile in water (with from 0 to 50% over 30min at 10 mL/min.

Example 16 Preparation of7-(2′-methyl-β-D-ribofuranosyl)-4-amino-5-(hydrazono)-pyrrolo[2,3-d]pyrimidine

To a solution of the compound from Step 5, Example 12 (20 mg, 0.05 mmol)in DMF was added hydrazine (2 μL, 0.060 mmol) and the reaction stirredat 50° C. for 2.5 hours. The crude reaction was purified directly onRP-HPLC on Phenominex column (250×20 mm) using a gradient ofacetonitrile in water (with from 0 to 50% over 30 min at 10 mL/min toyield 12 mg (75%) of the title compound;

¹H-NMR (CD₃OD): δ 8.03 (s, 1H), 7.85 (s, 1H), 7.68 (s, 1H), 6.21 (s,1H), 4.115-3.8 (m, 4H, sugar), 0.85 (s, 3H); MS: 323.14 (M+H).

Example 17 Synthesis of7-(2′-methyl-β-D-ribofuranosyl)-4-amino-5-(2-phenylethyn-1-yl)-pyrrolo[23-d]pyrimidine5′-triphosphate

7-(2′-methyl-β-D-ribofuranosyl)-4-amino-5-(2-phenylethyn-1-yl)-pyrrolo[2,3-d]pyrimidine5′-triphosphate (0.1 mmol) is co-evaporated 3 times with dry DMF,dissolved in 2 mL of PO(OMe)₃, cool to 5° C. and POCl₃ (35 μL) andproton sponge (64 mg) are added. The mixture is stirred at 5° C. for 3h, then tetrabutylammonium pyrophosphate (2 mmol, 4 mL of 0.5M solutionin DMF) is added and the mixture is kept for 2 more h at the sametemperature. The reaction is quenched with (Et₃N)HCO₃ buffer (pH 7.5)followed with water. The solvents are evaporated, the residue isdissolved in methanol (3 mL) and precipitated with ether (30 mL). Thesolid residue is purified by ion exchange HPLC on Vydac column (250×10mm) 0 to 100% B. Buffer A is 25 mM NaH₂PO₄/Na₂HPO₄, pH 3, buffer B is310 mM NaH₂PO₄/Na₂HPO₄, pH 3. The last peak is collected, concentratedup to the volume of 5 mL and repurified on RP HPLC on Phenominex column(250×20 mm) in gradient from 0 to 100% of buffer B in buffer A. Buffer Ais 0.5 M aqueous solution of triethylammonium acetate, buffer B is 0.5 Macetonitrile solution of triethylammonium acetate. Fractions containingthe title compound are combined, evaporated, co-evaporated 3 times withwater and lyophilized from water.

Example 18 Synthesis of7-(2′-methyl-β-D-ribofuranosyl)-4-amino-5-(2-phenylethyn-1-yl)-pyrrolo[23-d]pyrimidine5′-phosphate

7-(2′-methyl-β-D-ribofuranosyl)-4-amino-5-(2-phenylethyn-1-yl)-pyrrolo[2,3-d]pyrimidine(0.1 mmol) is co-evaporated 3 times with dry DMF, dissolved in 2 mL ofPO(OMe)₃, cool to 5° C. and POCl₃ (35 μL) and proton sponge (64 mg) areadded. The mixture is stirred at 5° C. for 3 h. The reaction is quenchedwith (Et₃N)HCO₃ buffer (pH 7.5) followed with water. The solvents areevaporated, the residue dissolved in methanol (3 mL) and precipitatedwith ether (30 mL). The solid residue is purified by ion exchange HPLCon Vydac column (250×10 mm) 0 to 100% B. Buffer A is 25 mMNaH₂PO₄/Na₂HPO₄, pH 3, buffer B is 310 mM NaH₂PO₄/Na₂HPO₄, pH 3. Thelast peak is collected, concentrated up to the volume of 5 mL andrepurified on RP HPLC on Phenominex column (250×20 mm) in gradient from0 to 100% of buffer B in buffer A. Buffer A is 0.5 M aqueous solution oftriethylammonium acetate, buffer B is 0.5 M acetonitrile solution oftriethylammonium acetate. Fractions containing the title compound arecombined, evaporated, co-evaporated 3 times with water and lyophilizedfrom water;

MS: 384.12 (M−H); ¹H-NMR: 7.97 & 7.67 (s, 1H each, base), 6.12 (s, 1H,H-1′), 4.10-3.85 (m, 4H, sugar), 3.15 (s, 1H, ethynyl), 0.87 (s, 3H,methyl-2′); ³¹P-NMR: 5.02 (s, 1P).

BIOLOGICAL EXAMPLES Example 1 Anti-Hepatitis C Activity

Compounds can exhibit anti-hepatitis C activity by inhibiting HCVpolymerase, by inhibiting other enzymes needed in the replication cycle,or by other pathways. A number of assays have been published to assessthese activities. A general method that assesses the gross increase ofHCV virus in culture is disclosed in U.S. Pat. No. 5,738,985 to Miles etal. In vitro assays have been reported in Ferrari et al. Jnl. of Vir.,73:1649-1654, 1999; Ishii etal., Hepatology, 29:1227-1235, 1999; Lohmannetal., Jnl of Bio. Chem., 274:10807-10815, 1999; and Yamashita et al.,Jnl. of Bio. Chem., 273:15479-15486, 1998.

WO 97/12033, filed on Sep. 27, 1996, by Emory University, listing C.Hagedom and A. Reinoldus as inventors, which claims priority to U.S.Provisional Patent Application.Ser. No. 60/004,383, filed on September1995, describes an HCV polymerase assay that can be used to evaluate theactivity of the of the compounds described herein. Another HCVpolymerase assay has been reported by Bartholomeusz, et al., Hepatitis CVirus (HCV) RNA polymerase assay using cloned HCV non-structuralproteins; Antiviral Therapy 1996: 1(Supp 4) 18-24.

Screens that measure reductions in kinase activity from HCV drugs aredisclosed in U.S. Pat. No. 6,030,785, to Katze et al., U.S. Pat. No.6,228,576, Delvecchio, and U.S. Pat. No. 5,759,795 to Jubin et al.Screens that measure the protease inhibiting activity of proposed HCVdrugs are disclosed in U.S. Pat. No. 5,861,267 to Su et al., U.S. Pat.No. 5,739,002 to De Francesco et al., and U.S. Pat. No. 5,597,691 toHoughton et al.

Example 2 Replicon Assay

A cell line, ET (Huh-lucubineo-ET) is used for screening of compounds ofthe present invention for HCV RNA dependent RNA polymerase. The ET cellline is stably transfected with RNA transcripts harboring aI₃₈₉luc-ubi-neo/NS3-3′/ET; replicon with fireflyluciferase-ubiquitin-neomycin phosphotransferase fusion protein andEMCV-IRES driven NS3-5B polyprotein containing the cell culture adaptivemutations (E1202G; T1280I; K1846T) (Krieger at al, 2001 andunpublished). The ET cells are grown in DMEM, supplemented with 10%fetal calf serum, 2 mM Glutamine, Penicillin (100 IU/mL)/Streptomycin(100 μg/mL), 1× nonessential amino acids, and 250 μg/mL G418(“Geneticin”). They are all available through Life Technologies(Bethesda, Md.). The cells are plated at 0.5-1.0×10⁴ cells/well in the96 well plates and incubated for 24 hrs before adding nucleosideanalogs. Then the compounds were added to the cells to achieve a finalconcentration of 50 or 100 μM,or whatever concentration is desired. Forthese determinations, 6 dilutions of each compound are used. Compoundsare typically diluted 3 fold to span a concentration range of 250 fold.Luciferase activity will be measured 48-72 hours later by adding a lysisbuffer and the substrate (Catalog number Glo-lysis buffer E2661 andBright-Glo leuciferase system E2620 Promega, Madison, Wis.). Cellsshould not be too confluent during the assay. Percent inhibition ofreplication will be plotted relative to no compound control. Under thesame condition, cytotoxicity of the compounds will be determined usingcell proliferation reagent, WST-1(Roche, Germany). IC₅₀ and TC₅₀ valuesare calculated by fitting % inhibition at each concentration to thefollowing equation, where b is Hill's coefficient:% inhibition=100%/[(IC50/[I])^(b)+1]

Example 3 Cloning and Expression of Recombinant HCV-NS5b

The coding sequence of NS5b protein is cloned by PCR frompFKI₃₈₉luc/NS3-3′/ET as described by Lohmann, V., et al. (1999) Science285, 110-113 using the following primers: (SEQ. ID. NO. 1)aggacatggatccgcggggtcgggcacgagacag (SEQ. ID. NO. 2)aaggctggcatgcactcaatgtcctacacatggac

The cloned fragment is missing the C terminus 21 amino acid residues.The cloned fragment is inserted into an IPTG-inducible expressionplasmid that provides an epitope tag (His)6 at the carboxy terminus ofthe protein.

The recombinant enzyme is expressed in XL-1 cells and after induction ofexpression, the protein is purified using affinity chromatography on anickel-NTA column. Storage condition is 10 mM Tris-HCl pH 7.5, 50 mMNaCl, 0.1 mM EDTA, 1 mM DTT, 20% glycerol at −20° C.

Example 4 HCV-NS5b Enzyme Assay

The polymerase activity is assayed by measuring incorporation ofradiolabeled UTP into a RNA product using a biotinylated,heteropolymeric template, which includes a portion of the HCV genome.Typically, the assay mixture (50 μL) contains 10 mM Tris-HCl (pH 7.5), 5mM MgCl₂, 0.2 mM EDTA, 10 mM KCl, 1 unit/μL RNAsin, 1 mM DTT, 10 μM eachof NTP, including [³H]-UTP, and 10 ng/μL heteropolymeric template. Testcompounds are initially dissolved in 100% DMSO and further diluted inaqueous buffer containing 5% DMSO. Typically, compounds are tested atconcentrations between 1 nM and 100 μM. Reactions are started withaddition of enzyme and allowed to continue at 37° C. for 2 hours.Reactions are quenched with 8 μL of 100 mM EDTA and reaction mixtures(30 μL) are transferred to streptavidin-coated scintillation proximitymicrotiter plates (FlashPlates) and incubated at 4° C. overnight.Incorporation of radioactivity is determined by scintillation counting.

Shown below in Table II are the results for the replicon assay or theenzyme assay used to test the compounds of this invention. TABLE IIReplicon, Enzyme, % inhibition % inhibition Cmpd # Structure @ 100 μM @50 μM @ Con 1 @ Con 2 101

98.5 98.1 105

89.1 69.6 106

95.6 95.5 133

96.7 92.3 137

97.6 98.1 145

91.8 90.7 154

19.2 @ 50 μM 10.9 @ 16.7 μM 157

95   89 158

cis and trans isomer tested 78.6 62.4 75.6 35.8 165

95.9 96.8 166

not tested at 100 μM second test 96.6 @ 16.7 μM 97 168

96 @ 23.1 μM 92 @ 7.7 μM 169

89.6 80.7 173

86.9 76.7 181

98.1 98.2

FORMULATION EXAMPLES

The following are representative pharmaceutical formulations containinga compound of the present invention.

Example 1 Tablet Formulation

Ingredient Quantity per tablet, mg compound of this invention 400cornstarch 50 croscarmellose sodium 25 lactose 120 magnesium stearate 5

Example 2 Capsule Formulation

The following ingredients are mixed intimately and loaded into ahard-shell gelatin capsule. Ingredient Quantity per capsule, mg compoundof this invention 200 lactose, spray-dried 148 magnesium stearate 2

Example 3 Suspension Formulation

The following ingredients are mixed to form a suspension for oraladministration. Ingredient Amount compound of this invention 1.0 gfumaric acid 0.5 g sodium chloride 2.0 g methyl paraben 0.15 g propylparaben 0.05 g granulated sugar 25.0 g sorbitol (70% solution) 13.00 gVeegum K (Vanderbilt Co.) 1.0 g flavoring 0.035 mL colorings 0.5 mgdistilled water q.s. to 100 mL

Example 4 Injectable Formulation

The following ingredients are mixed to form an injectable formulation.Ingredient Amount compound of this invention 0.2 mg-20 mg sodium acetatebuffer solution, 0.4 M 2.0 mL HCl (1N) or NaOH (1N) q.s. to suitable pHwater (distilled, sterile) q.s. to 20 mL

Example 5 Suppository Formulation

A suppository of total weight 2.5 g is prepared by mixing the compoundof the invention with Witepsol® H-15 (triglycerides of saturatedvegetable fatty acid; Riches-Nelson, Inc., New York), and has thefollowing composition: Ingredient Amount compound of the invention 500mg Witepsol ® H-15 balance

1-45. (canceled)
 46. A method for treating a viral infection in a mammalmediated at least in part by a virus in the flaviviridae family ofviruses which method comprises administering to a mammal, that has beendiagnosed with said viral infection or is at risk of developing saidviral infection, a compound of Formula I:

wherein Y is selected from the group consisting of a bond, —CH₂— or —O—;each of W, W¹ and W² is independently selected from the group consistingof hydrogen, acyl, oxyacyl, phosphonate, phosphate esters, phosphate,phosphonamidate, phosphorodiamidate, phosphoramidate monoester, cyclicphosphoramidate, cyclic phosphorodiamidate, phosphoramidate diester, and—C(O)CHR³⁰NH₂ where R³⁰ is selected from the group consisting ofhydrogen, alkyl, substituted alkyl aryl, substituted aryl, heteroaryl,substituted heteroaryl, and a sidechain of an amino acid; and T isselected from the group consisting of a) —-C≡C—R, where R is selectedfrom the group consisting of i) tri(C₁-C₄)alkylsilyl, —C(O)NR¹R²,alkoxyalkyl, heteroaryl, and substituted heteroaryl, and phenylsubstituted with 1 to 3 substituents selected from the group consistingof alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, aminoacyl, amidino, amino, substituted amino, carboxyl,carboxyl ester, cyano, cycloalkyl, substituted cycloalkyl, cycloalkoxy,substituted cycloalkoxy, guanidino, halo, heteroaryl substitutedheteroaryl, hydrazino, hydroxyl, nitro, thiol, and —S(O)_(m)R³; where R¹and R² are independently selected from the group consisting of hydrogen,alkyl, substituted alkyl, amino, substituted amino, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclyl and substitutedheterocyclyl provided that only one of R¹ and R² is amino or substitutedamino, and further wherein R¹ and R², together with the nitrogen atompendant thereto, form a heterocyclyl or substituted heterocyclyl; R³ isselected from the group consisting of alkyl, substituted alkyl, amino,substituted amino, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclyl and substituted heterocyclyl; and m is aninteger equal to 0, 1 or 2; ii) —C(O)OR¹⁴, where R¹⁴ is hydrogen, alkylor substituted alkyl; b) —C(O)H; c) —CH═NNHR¹⁵, where R¹⁵ is H or alkyl;d) —CH═N(OR¹⁵), where R¹⁵ is as defined above; e) —CH(OR¹⁶)₂ where R¹⁶is (C₃-C₆)alkyl and f) —B(OR¹⁵)₂ where R¹⁵ is as defined above; andpharmaceutically acceptable salts or partial salts thereof, whereinsubstituted alkyl refers to an alkyl group having from 1 to 3substituents selected from the group consisting of alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, oxyacyl, amino, substituted amino,aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano,halogen, hydroxyl, nitro, carboxyl, carboxyl esters, cycloalkyl,substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclyl, and substituted heterocyclyl; alkoxy refers to alkyl-O—;substituted alkoxy refers to (substituted alkyl)-O—; alkoxyalkyl refersto -alkylene(alkoxy)_(n) or -alkylene(substituted alkoxy)_(n), wherealkylene is a divalent straight or branched chain alkylene group of from1 to 3 carbon atoms; acyl refers to a moiety selected from the groupconsisting of alkyl-C(O)—, substituted alkyl-C(O)—, alkenyl-C(O)—,substituted alkenyl-C(O)—, alkynyl-C(O)—, substituted alkynl-C(O)—cycloalkyl-C(O)—, substituted cycloalkyl-C(O)—, aryl-C(O)—, substitutedaryl-C(O)—, heteroaryl-C(O)—, substituted heteroaryl-C(O),heterocyclyl-C(O)—, and substituted heterocyclyl-C(O)—; acylamino refersto the group —C(O)NR⁴R⁴ where each R⁴ is independently selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substitutedheteroaryl, heterocyclyl, substituted heterocyclyl and where each R⁴ isjoined to form together with the nitrogen atom a heterocyclyl orsubstituted heterocyclyl ring; acyloxy refers to a moiety selected fromthe group consisting of alkyl-C(O)O—, substituted alkyl-C(O)O—,alkenyl-C(O)O—, substituted alkenyl-C(O)O—, alkynyl-C(O)O—, substitutedalkynyl-C(O)O—, aryl-C(O)O—, substituted aryl-C(O)O—, cycloalkyl-C(O)O—,substituted cycloalkyl-C(O)O—, heteroaryl-C(O)O—, substitutedheteroaryl-C(O)O—, heterocyclyl-C(O)O—, and substitutedheterocyclyl-C(O)O—; oxyacyl refers to a moiety selected from the groupconsisting of alkyl-OC(O)—, substituted alkyl-OC(O)—, alkenyl-OC(O)—,substituted alkenyl-OC(O)—, alkynyl-OC(O)—, substituted alkynyl-OC(O)—,aryl-OC(O)—, substituted aryl-OC(O)—, cycloalkyl-OC(O)—, substitutedcycloalkyl-OC(O)—, heteroaryl-OC(O)—, substituted heteroaryl-OC(O)—,heterocyclyl-OC(O)—, and substituted heterocyclyl-OC(O)—; substitutedalkenyl refers to an alkenyl group having from 1 to 3 substituentsselected from the group consisting of alkoxy, substituted alkoxy, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aryl,substituted aryl, aryloxy, substituted aryloxy, cyano, halogen,hydroxyl, nitro, carboxyl, carboxyl esters, cycloalkyl, substitutedcycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl, andsubstituted heterocyclyl with the proviso that any hydroxyl substitutionis not attached to an unsaturated carbon atom; substituted alkynylrefers to an alkynyl group having from 1 to 3 substituents selected fromthe group consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl,aryloxy, substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl,carboxyl esters, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclyl, and substituted heterocyclyl;amino refers to —NH₂. substituted amino refers to —NR′R″, where R′ andR″ independently are selected from the group consisting of hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkenyl,substituted alkynyl, aryl, substituted aryl, cycloalkyl, substitutedcycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl,substituted heterocyclyl and where R′ and R″ are joined, together withthe nitrogen bound thereto to form a heterocyclyl or substitutedheterocyclyl group provided that R′ and R″ are both not hydrogen;amidino refers to —C(═NR¹¹)NR¹¹R¹¹ where each R¹¹ independently ishydrogen or alkyl; aminoacyl refers to a moiety selected from the groupconsisting of —NR⁵C(O)alkyl, —NR⁵C(O)substituted alkyl,—NR⁵C(O)cycloalkyl, —NR⁵C(O)substituted cycloalkyl, —NR⁵C(O)alkenyl,—NR⁵C(O)substituted alkenyl, —NR⁵C(O)alkynyl, —NR⁵C(O)substitutedalkynyl, —NR⁵C(O)aryl, —NR⁵C(O)substituted aryl, —NR⁵C(O)heteroaryl,—NR⁵C(O)substituted heteroaryl, —NR⁵C(O)heterocyclyl, and—NR⁵C(O)substituted heterocyclyl where R⁵ is hydrogen or alkyl; arylrefers to a monovalent aromatic carbocyclic group of from 6 to 14 carbonatoms having a single ring (e.g., phenyl) or multiple condensed rings(e.g., naphthyl or anthryl) which condensed rings may or may not bearomatic (e.g., 2-benzoxazolinone, 2H-1 4-benzoxazin-3(4H)-one-7-yl, andthe like) provided that the point of attachment is at an aromatic carbonatom; substituted aryl refers to an aryl group which is substituted withfrom 1 to 3 substituents selected from the group consisting of hydroxyl,acyl, acylamino, acyloxy, alkyl, substituted alkyl, alkoxy, substitutedalkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,amino, substituted amino, aminoacyl, aryl, substituted aryl, aryloxy,substituted aryloxy, cycloalkoxy, substituted cycloalkoxy, carboxyl,carboxyl esters, cyano, thiol, thioalkyl, substituted thioalkyl,thioaryl, substituted thioaryl, thioheteroaryl, substitutedthioheteroaryl, thiocycloalkyl, substituted thiocycloalkyl,thioheterocyclyl, substituted thioheterocyclyl, cycloalkyl, substitutedcycloalkyL, halo, nitro, heteroaryl, substituted heteroaryl,heterocyclyl, substituted heterocyclyl, heteroaryloxy, substitutedheteroaryloxy, heterocyclyloxy, and substituted heterocyclyloxy; aryloxyrefers to the group aryl-O—; substituted aryloxy refers to (substitutedaryl)-O—; carboxyl refers to —COOH or salts thereof; carboxyl esterrefers to a moiety selected from the group consisting of —C(O)O-alkyl,—C(O)O-(substituted alkyl), —C(O)O-aryl, and —C(O)O-(substituted aryl);cycloalkyl refers to a cyclic alkyl group of from 3 to 10 carbon atomshaving single or multiple cyclic rings; substituted cycloalkyl refers toa cycloalkyl having from 1 to 5 substituents selected from the groupconsisting of oxo (═O), thioxo (═S), alkyl, substituted alkyl, alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano,halogen, hydroxyl, nitro, carboxyl, carboxyl esters, cycloalkyl,substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclyl, and substituted heterocyclyl; cycloalkoxy refers to—O-cycloalkyl, substituted cycloalkoxy refers to —O-(substitutedcycloalkyl); guanidino refers to —NR¹²C(═NR¹²)NR¹²R¹² where each R¹²independently is hydrogen or alkyl; halogen refers to fluoro, chloro,bromo and iodo; heteroaryl refers to an aromatic group of from 1 to 10carbon atoms and 1 to 4 heteroatoms selected from the group consistingof oxygen, nitrogen, and sulfur within the ring wherein the nitrogenand/or sulfur is optionally oxidized ((N→O) —S(O)—, or —SO₂—), whereinthe heteroaryl group can have a single ring or multiple condensed ringswhere the condensed rings may or may not be aromatic and/or contain aheteroatom provided that the point of attachment is through an aromaticring atom; substituted heteroaryl refers to a heteroaryl group that issubstituted with from 1 to 3 substituents selected from the groupconsisting of hydroxyl, acyl, acylamino, acyloxy, alkyl substitutedalkyl alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, amino, substituted amino, aminoacyl, aryl,substituted aryl, aryloxy, substituted aryloxy, cycloalkoxy, substitutedcycloalkoxy, carboxyl carboxyl esters, cyano, thiol thioalkylsubstituted thioalkyl, thioaryl, substituted thioaryl, thioheteroaryl,substituted thioheteroaryl, thiocycloalkyl, substituted thiocycloalkyl,thioheterocyclyl, substituted thioheterocyclyl, cycloalkyl, substitutedcycloalkyl, halo, nitro, heteroaryl, substituted heteroaryl,heterocyclyl, substituted heterocyclyl, heteroaryloxy, substitutedheteroaryloxy, heterocyclyloxy, and substituted heterocyclyloxy;heteroaryloxy refers to the group —O-heteroaryl; substitutedheteroaryloxy refers to the group —O-(substituted heteroaryl);heterocyclyl refers to a saturated or unsaturated group, but notheteroaryl, having a single ring or multiple condensed rings, from 1 to10 carbon atoms and from 1 to 4 hetero atoms selected from the groupconsisting of nitrogen, oxygen and sulfur within the ring wherein thenitrogen and/or sulfur atoms can be optionally oxidized ((N→O), —S(O)—or —SO₂—) and further wherein, in fused ring systems, one or more therings can be cycloalkyl, aryl or heteroaryl provided that the point ofattachment is through the heterocyclyl ring; substituted heterocyclylrefers to a heterocycle group that is substituted with from 1 to 3substituents selected from the group consisting of oxo (═O), thioxo(═S), alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl aryl,substituted aryl, aryloxy, substituted aryloxy, cyano, halogen,hydroxyl, nitro, carboxyl, carboxyl esters, cycloalkyl, substitutedcycloalkyl, heteroaryl, substituted heteroary, heterocyclyl, andsubstituted heterocyclyl; heterocyclyloxy refers to the group—O-heterocyclyl; substituted heterocyclyloxy refers to the group—O-(substituted heterocyclyl; hydrazino refers to the group —NR¹³NR¹³R¹³wherein each R¹ ³ independently is hydrogen or alkyl; phosphate refersto the groups —OP(O)(OH)₂ (monophosphate), —OP(O)(OH)OP(O)(OH)₂(diphosphate) and —OP(O)(OH)OP(O)(OH)OP(O)(OH)₂ (triphosphate) or saltsthereof including partial salts thereof; phosphate ester refers to amono-, di- or tri-phosphate, wherein one or more of the hydroxyl groupis replaced by an alkoxy group; phosphonate refers to a moiety selectedfrom the group consisting of —OP(O)(R⁶)(OH) and —OP(O)(R⁶)(OR⁶) andsalts thereof, including partial salts thereof, wherein each R⁶ isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl carboxylic acid, and carboxyl ester;phosphorodiamidate refers to

 where each R⁷ may be the same or different and each is hydrogen, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl;phosphoramidate monoester refers to

 where R³⁰ is selected from the group consisting of hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, and a sidechain of an aminoacid and R⁸ is hydrogen or alkyl; phosphoramidate diester refers to

 where R³⁰ is selected from the group consisting of hydrogen, alkyl,substituted alkyl aryl substituted aryl and a sidechain of an aminoacid, R⁸ is hydrogen or alkyl and R¹⁰ is selected from the groupconsisting of alkyl, substutituted alkyl, aryl, substituted aryl,cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclyl and substituted heterocyclyl; cyclic phosphoramidate refersto

 where n is 1 to 3; cyclic iphosphorodiamidate refers to

 where n is 1 to 3; phosphonamidate refers

 where R¹⁴ is hydrogen, alkyl, substituted alkyl, cycloalkyl, orsubstituted cycloalkyl; thiol refers to —SH; thioalkyl refers to—S-alkyl; substituted thioalkyl refers to —S-(substituted alkyl);thiocycloalkyl refers to —S-cycloalkyl; substituted thiocycloalkylrefers to —S-(substituted cycloalkyl); thioaryl refers to —S-aryl;substituted thioaryl refers to —S-(substituted aryl); thioheteroarmlrefers to —S-heteroaryl; substituted thioheteroaryl refers to—S-(substituted heteroaryl); thioheterocyclyl refers to —S-heterocyclyl;and substituted thioheterocyclyl refers to —S-(substitutedheterocyclyl).
 47. (canceled)
 48. The method of claim 46, wherein theviral infection is mediated at least in part by a hepatitis C virus(HCV) and wherein the method further comprises administration of atherapeutically effective amount of one or more agents active againstHCV in combination with the compound.
 49. The method of claim 48 whereinsaid active agent against is Ribavirin, levovirin, viramidine, thymosinalpha-1, an inhibitor of NS3 serine protease, and inhibitor of inosinemonophosphate dehydrogenase, interferon-alpha, pegylatedinterferon-alpha, alone or in combination with Ribavirin or levovirin.50. The method of claim 49 wherein said agent active against HCV isinterferon-alpha or pegylated interferon-alpha alone or in combinationwith Ribavirin or levovirin.